Mapping developmental and targeted secretory signatures of euploid and aneuploid human embryos using an advanced...

Do euploid (EU), trisomy 21 (T21), and monosomy 21 (M21) human embryos exhibit different patterns of secreted proteins and morphometric changes during extended in vitro culture, either with or without endometrial cell support?

Secreted protein profiles and morphometric parameters discriminate between euploid and aneuploid embryos during post-implantation in vitro culture with endometrial cell support.

Extended in vitro culture systems with and without endometrial cells have enabled the study of early human post-implantation development and highlighted the importance of embryo-endometrial crosstalk during implantation. However, how embryo chromosomal status influences implantation dynamics and embryo-endometrial interactions remain poorly understood.

Laboratory-based study. Sixty-nine supernumerary human embryos donated for research at blastocyst stage (5 days post-fertilization, d.p.f.) (EU, n = 24; T21, n = 21; M21, n = 24) were used.

Extended in vitro culture until 14 d.p.f. was performed using two systems: a cell-free culture medium optimized for post-implantation development, and a 3D culture based on cell-engineered receptive endometrial scaffold technology (CREST), providing epithelial and stromal cell support. Secreted levels of CALR (calreticulin), β-hCG, human placental lactogen (hPL), pregnancy-associated plasma protein A (PAPP-A), and placental growth factor (PlGF) were quantified at different developmental time points corresponding to 7, 10, 12, and 14 d.p.f. Embryo morphometric parameters, including area, degree of expansion, axis ratio, and circularity were also measured.

Embryos cultured without endometrial support showed compromised viability beyond 10 d.p.f., with significantly reduced secretion of β-hCG, hPL, PAPP-A, PlGF, and CALR. The area of post-implanted EU and T21 embryos cultured in cell-free medium progressively increased up to 9 d.p.f., unlike M21 embryos. However, all showed no changes in blastocoel expansion and maintained a rounded morphology throughout all post-implantation culture. CREST systems extended viability up to 14 d.p.f. and enhanced secretion levels of all evaluated protein markers compared with embryos cultured in cell-free conditions. Among embryos cultured in CREST systems, M21 embryos exhibited diminished hPL, CALR, and PlGF secretion as early as 7 d.p.f. At 10 d.p.f., PAPP-A levels distinguished EU from both T21 and M21 embryos. Progressive increases in PAPP-A and PlGF were observed in EU embryos until 14 d.p.f., but not in T21 embryos. Morphometric analysis revealed that only EU and T21 embryos underwent implantation-like expansion, blastocoel collapse, and increased axis ratio. In contrast, M21 embryos retained a rounded morphology and failed to expand.

No OMIC technique was performed in this research.

One of the main limitations of this study is the number of embryos used. Although more than 10 embryos were included in each culture condition, biological variability may still have influenced the results. This limitation is related to the difficulty of obtaining pure chromosome 21 aneuploid embryos. In addition, while the CREST system successfully recapitulates key structural features of the endometrium, it lacks endothelial and immune cell components, which may influence embryo development and implantation dynamics. Finally, we selected a targeted set of secreted proteins based on their relevance to the main objective of the study; however, the use of broader secretomic approaches could have provided a more comprehensive characterization of embryo-endometrial interactions. The findings are limited to pure chromosome 21 aneuploidies; therefore, further studies including aneuploidies affecting other chromosomes are warranted to assess the generalizability of the results.

The obtained results demonstrate the usefulness of the CREST system to study implantation process. Besides, these findings support the potential of secreted proteins analysis in extended culture as a non-invasive strategy to improve embryo selection beyond conventional morphological assessment. In parallel, we establish the CREST model as a translational platform to identify novel reproductive competence biomarkers and evaluate interventions aimed at improving maternal-embryo communication, not only after implantation but also during the pre-implantation phase.

This research was funded by the Institute of Health Carlos III (ISCIII) (Spanish Ministry of Science and Innovation) through the FIS program granted to F.D. (PI20/00405, PI23/00860), co-funded by the European Union, the Generalitat Valenciana through the Grant Program for Consolidated Research Groups (CIAICO/2022/203 [F.D.]), Centro para el Desarrollo Tecnológico y la Innovación (CDTI; reference number IDI-20210916) (Spanish Ministry of Science and Innovation) granted to I.S.-R. and Fundación Merck Salud through XXXI Merck Research Grant on Fertility granted to F.D. A.P. and R.Y.-S. are funded by Generalitat Valenciana Grant for the hiring of support staff linked to a technology transfer project (CIAPOT/2022/18 [A.P.]) and training research (CIACIF/2021/051 [R.Y.-S.]). S.M.-A. is funded by Institute of Health Carlos III (ISCIII) (Spanish Ministry of Science and Innovation) through the contract of training research personnel into health (FI24/00068), co-funded by the European Union. M.A.M. was supported by the Stanford Dunlevie Maternal Fetal Medicine Center for Discovery, Innovation and Impact, the Duan Family Faculty Scholar in Maternal-Fetal Medicine, the FY25 ASRM Discovery & Innovation Grant from the American Society for Reproductive Medicine, and the California Institute for Regenerative Medicine (DISC0-17685).

Matteo Molè, declares that he serves as an advisory board member and consultant for Opal Therapeutics (CA, USA) and Genie Fertility (UK). He is also a co-inventor of the patent 'A Method of Generating an In Vitro Endometrium Assembloid' (UK Patent No. BAB-C-P3371GBp). All other authors have no financial nor competing interest to declare.

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