Utilization of a three-dimensional in vitro co-culture system to characterize embryonic mechanisms associated with implantation

OBJECTIVE: Implantation success is dependent on timely molecular signaling to establish embryonic apposition, adhesion, and invasion. In an effort to elucidate this critical period in human reproduction, the objective of this study was to use a novel, time-lapse three-dimensional (3D) in vitro co-culture system to characterize the timing of blastocyst development on the initial stages of implantation. DESIGN: Endometrial biopsies were collected from fertile oocyte donors to generate individual 3D wells of separated monolayers of luminal endometrial epithelial and stromal cells for co-culture with an individual blastocyst (n = 72; maternal age = 36.3 ± 5.1 years). After 72 hours of co-culture (CytoSMART Lux3 time-lapse imaging system), blastocysts were evaluated for stage of implantation and separated into two groups: No implantation (no adhesion or invasion) and implantation (complete adhesion and invasion). SUBJECTS: N/A. EXPOSURE: N/A. MAIN OUTCOME MEASURES: Immunohistochemistry and targeted quantitative real-time polymerase chain reaction gene expression were performed on individual blastocysts and on exosome-purified small RNAs derived from supernatant. RESULTS: After successful implantation into the endometrial epithelium, correctly timed blastocysts experienced greater duration of apposition and adhesion, delayed onset of invasion, and increased number of spontaneous blastocyst collapse compared with slower developing blastocysts. Additionally, targeted gene expression analysis revealed an upward trend of implantation-promoting genes GATA binding protein 3, octamer binding transcription factor 4, and cell death regulatory gene caspase protein 3 in correctly timed blastocysts compared with slower developing blastocysts. Interestingly, as blastocysts became more attached to the epithelium, a downward trend of developmental genes caudal-related homeobox 2 and bone morphogenic protein 15 was observed. A downward trend of hsa-miR-1-3p and an upward trend of hsa-miR-34b-5p was observed in the supernatant of co-cultured blastocysts that achieved successful implantation in co-culture. Top Kyoto Encyclopedia of Genes and Genomes pathways impacted by these microRNAs were axon guidance, ubiquitin-mediated proteolysis, and neurotrophin signaling pathway. CONCLUSION: Time-lapse 3D in vitro co-culture revealed that the timing of blastocyst development is critical to the initial stages of implantation. The ability of the trophectoderm to expand, orient, and initiate apposition may contribute to the higher likelihood of success as indicated by altered gene expression and regulatory pathways.