Fig 1: Schematic overview of the protocol. The workflow includes; a, initial PXGL hPSCs seeding and passaging (Steps 1-25), b,procedure for the formation of human blastoid from hPSCs cultured in PXGL medium (Steps 25-65) and c, Seeding and passaging endometrial organoids (Steps 66-103), OFEL formation (Steps 104-125) and Implantation assay (Steps 126-138) a, Human embryonic stem cells (ESCs) or induced PSCs (iPSCs) cultured in PXGL medium are seeded into feeder-layers for maintenance and passaged several times before human blastoid formation (Steps 1-25). b, hPSCs are dissociated into single cells (Steps 35-47) and plated in non-adherent hydrogel microwells or AggreWell plate to form small aggregates of the cells (steps 48-53). Upon exposure to lysophosphatidic acid (LPA), A83-01 and PD0325901 in a chemically defined medium containing leukaemia inhibitory factor (LIF) and Y-27632 for 2 days, some aggregates form small cavities (steps 54-61). With continuing culture for 2 days in a medium containing LPA and Y-27632, human blastoidss are formed efficiently and consistently (Steps 62-65). N2B27, serum-free medium; PALLY, PD0325901 + A83-01 + LPA + hLIF + Y-27632; LY, LPA + Y-27632. c, Procedure for the formation of open faced endometrial layer (OFEL) from human endometrial organoids. Human endometrial organoids are seeded and passaged before OFEL formation (Steps 66-103) and then are dissociated into single cells and small colonies and plated in Matrigel-coated wells (Steps 104-120). After 1-2 days, the cells become confluent and grow as a monolayer. Once confluency is reached, E2 supplemented medium is added to the cell for 2 days (Steps 121-122) and then, OFELs are stimulated with E2, P4, cAMP, and XAV-939 for 4 days to make them ready for implantation assay (Steps 123-125). In the next step, human blastoids that display blastocyst morphology with compact ICM are selected and transferred onto OFELs (Steps 126-135). Attachment and development of blastoids will be monitored in the next days (Step 136). OFEL, open face endometrial layer. EPC, E2+P4+cAMP.