Utilization of a 3D in vitro co-culture system to characterize embryonic mechanisms associated with implantation.

Abstract

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 utilize a novel, time-lapse 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). Following 72h 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 Intervention (for RCT) or Exposure (for observational studies): N/A MAIN OUTCOME MEASURES: Immunohistochemistry and targeted qPCR gene expression were performed on individual blastocysts and on exosome purified small RNAs derived from supernatant.

Results: Following 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 to slower developing blastocysts. Additionally, targeted gene expression analysis revealed an upward trend of implantation promoting genes GATA3, OCT4, and cell death regulatory gene CASP3 in correctly timed compared to slower developing blastocysts. Interestingly, as blastocysts became more attached to the epithelium, a downward trend of developmental genes CDX2 and BMP15 was observed. A downward trend of hsa-miR-1-3p and upward trend of hsa-miR-34b-5p was observed in the supernatant of co-cultured blastocysts that achieved successful implantation in co-culture. Top KEGG pathways impacted by these microRNA's 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.