Comparative analysis of the transcriptome and methylome in distinct trophoblast populations at the time of embryo implantation in mares

During equine implantation, two distinct trophoblast subpopulations are identified: the invasive chorionic girdle (CG) and the non-invasive allantochorion. The invasive CG trophoblast is believed to modulate the maternal immune response and facilitates embryo attachment to the endometrium, while the allantochorion contributes to placental development without breaching the endometrium. However, the mechanisms underlying differential adhesion and immune modulation by these two lineages remain poorly understood. This study aimed to investigate the regulation of biological processes underlying trophoblast attachment, development and maintenance of immune tolerance by comparing the transcriptomes and methylomes of CG and allantochorion trophoblasts before and after implantation, that is, between gestational days 33 and 42. Irrespective of gestational day, allantochorion (n = 4) was enriched in TGFB and its receptors TGFBR1 and TGFBR2. Across the implantation window, expression of regulatory macrophage markers such as IL33, PLG-RKT, and CD163 increased in the allantochorion, along with upregulation of TFEB and SERPINB9. The invasive CG (CG33, n = 4) showed higher expression of NLRC5, a transcriptional regulator of MHC I, compared to the non-invasive allantochorion (ALC33, n = 4). Notably, NLRC5 expression declined in the allantochorion from day 33 to 42. Furthermore, Beta-2 microglobulin (B2M), which encodes the MHC I light chain, was hypermethylated in the allantochorion. Several genes, including CAVIN1, LHB, and INSR, were both differentially expressed and methylated between the two trophoblast types, while others, such as IFNGR1, CD177, showed differential regulation across the implantation window. Gene Ontology and pathway analyses revealed that differentially expressed and methylated genes were enriched in biological processes and pathways critical for implantation, including cell migration and adhesion, TGFB signaling, fibrosis, wound healing, and leukocyte extravasation. The increased expression of IL33, and CD163 suggests that the allantochorion may promote the recruitment or polarization of macrophages toward a regulatory phenotype, thereby modulating the local immune environment. TFEB may contribute to the steroidogenic activity and functional maturation of the allantochorion. Spatial and temporal differences in gene methylation between CG and allantochorion suggest involvement of epigenetic regulation in trophoblast invasion and attachment to the endometrium. Finally, combined B2M hypermethylation and differential expression of NLRC5 point to epigenetic and transcriptional control of MHC I expression in the equine trophoblasts during implantation.