(173) Investigating Implantation Dynamics through Aneuploid Embryos with Distinct Implantation Potentials

Heather M. Rogers^1,2, Maria Carolina Zimara¹, William B. Schoolcraft¹, Mandy Katz-Jaffe¹, Ye Yuan¹

1. Colorado Center for Reproductive Medicine, Lone Tree, Colorado, USA

2. Colorado State University, Fort Collins, Colorado, USA

 

Abstract Text:

Embryo aneuploidy is a leading cause of implantation failure and miscarriage in human reproduction. This study aims to characterize embryos with varying chromosomal trisomies beyond the blastocyst stage to uncover mechanisms of how genetic aneuploidies influence post-implantation human development. A total of 184 good quality, PGT-A tested human blastocysts donated for research with IRB approval, were included in this study. Embryos were categorized into four groups based on published implantation potential: 1) euploid, 2) implantation failure (Trisomies 1 and 19), 3) high implantation followed by fetal demise (Trisomies 16 and 22), and 4) live birth potential but majority fetal demise (Trisomies 13 and 18). Embryos were warmed using standard protocols, and placed into extended embryo culture (EEC) on fibronectin-coated wells for 5 days. Stereoscope images were taken on EEC day 3 to 5 to evaluate trophoblast outgrowth, and spent media were collected on EEC day 5 for hCG quantification. After 5 days, embryos were either processed for immunofluorescence imaging (DAPI, POUF51, and SOX17) to assess epiblast and primitive endoderm (PE) development, or dissociated for single-cell RNA-sequencing using BIORAD’s ddSEQ scRNA library prep kit. Numerical variables are displayed as mean±SEM and were analyzed using a one-way ANOVA (p< 0.05). On EEC day 4, Trisomy 16 embryos exhibited significantly larger outgrowth areas than Trisomies 19 and 22 (1.30±0.12, 0.66±0.09, and 0.69±0.10 in mm², respectively), indicating enhanced trophoblast proliferation. This difference became even more pronounced by EEC day 5, with Trisomy 16 outgrowths exceeding those of Trisomies 1, 19, and 22 (2.49±0.30, 1.16±0.16, 1.01±0.14, and 0.99±0.30 in mm², respectively). The high implantation followed by fetal demise group (Trisomies 16 and 22) also displayed significantly more SOX17 positive cells compared to implantation failure (Trisomies 1 and 19) and live birth potential but majority fetal demise (Trisomies 13 and 18) groups, suggestion impaired PE development in the latter groups. The implantation failure group (Trisomies 1 and 19) had significantly less hCG production compared to the euploid and live birth potential but majority fetal demise (Trisomies 13 and 18) groups (7905±2222, 13923±1949, 13809±1994 mIU/mL, respectively), indicating impaired syncytiotrophoblast development in the implantation failure group. No significant differences were observed among groups in the presence of epiblasts, epiblast cell number, and total cell number. Trisomy 16 embryos, classified as high implantation potential followed by fetal demise, demonstrated superior trophoblast outgrowth compared to embryos in the implantation failure group, supporting their potential for early implantation success. In contrast, Trisomy 1 and 19 embryos, classified as implantation failures, demonstrated a lack of PE development, hypoproliferation of the trophoblast, and impaired differentiation of hCG-producing syncytiotrophoblast cells. Ongoing single-cell RNA sequencing analyses will provide deeper insights into key molecular pathways driving implantation success or failure among human embryos with varying developmental potential. This study explored the mechanisms underlying the developmental failure of aneuploid embryos, providing insights that may inform potential interventions to support euploid embryos during in vitro culture and embryo transfer, with the ultimate goal of enhancing implantation potential and improving patient outcomes.