Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid–endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri-and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo–endometrium crosstalk.

The authors have declared no competing interest.

This work was supported by the Estonian Research Council grants (PSG1082 and PRG1076); Centre for Innovative Medicine (CIMED), Region Stockholm no. FoUI-963306; Swedish Research Council grant no. 2024-02530 and Novo Nordisk Foundation grant no. NNF24OC0092384. DL was supported by the internal financing from the Institute of Chemistry, University of Tartu, Estonia (PLTKTARENG21). A.S. was supported by Horizon Europe (NESTOR, grant no. 101120075) and the Estonian Ministry of Education and Research Centres of Excellence grant TK214 name of CoE. HK was supported by the Sigrid Juselius Foundation. MB was supported by Wenner-Gren foundation (grant no. UPD2022-0108). KH was supported by Estonian Research Council (PUTJD1253). PD and IR received financial support from the European Union (ERC, SAFER, 101124440). Views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics Committee of the University of Tartu gave ethical approval for this work (approval numbers 330/M-8, issued 16 October 2020, and 364/M-9, issued 16 May 2022) for the collection and use of endometrial biopsies from healthy female donors at South Estonian Hospital (Voru, Estonia). Ethics Committee of Karolinska Institutet gave ethical approval for this work (permit numbers EPN #454/02 and #2011/745/31-3) for the use of human embryonic stem cells for the generation of blastoids and their use in co-culture experiments.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The mRNA sequencing raw data are available are available from the corresponding author upon request.