P-136 Cell exclusion during human embryo development results in altered morphokinetic patterns up to morula formation
Authors: Kakulavarapu R , Stensen M.H , Haugen T.B , Delbarre E 
Published: June 2022

Abstract

Study question
Do embryos that present with cell exclusion show dysregulated cleavage divisions and deviating reproductive success, compared to normally dividing embryos?

Summary answer
Exclusion embryos have lower reproductive success, showing patterns of delayed and rapid cleavage until morulation, suggesting potential compensatory mechanisms and cellular checkpoints in post-compaction stages

What is known already
Within assisted reproductive technology, visual observation of embryo development constitutes a major contribution to assessments of embryo competence. Time-lapse technology has allowed for detailed identification and measurement of dynamic events that impact embryo development and reproductive outcome. Two recent studies have identified an anomalous pattern of cell division occurring during early embryo development, termed cell exclusion. These cells seem to arrest during early cleavage stages and fail to participate in downstream compaction, thereby resulting in lower reproductive success. Previous research suggests aberrant chromosomal re-arrangements or irregular cellular content may contribute to negatively associated reproductive outcomes.

Study design, size, duration
This study is based on a single-clinic retrospective analysis of 515 time-lapse videos collected from 2013-2019. Embryos were cultured in EmbryoScope incubators for 4-5 days post-insemination by conventional in vitro fertilization or intracytoplasmic sperm injection, followed by fresh transfer. Supervised annotations were made for cell divisions and morphological quality by three embryologists. An in-depth morphokinetic analysis was conducted using student’s t-test and analysis of variance for continuous variables.

Participants/materials, setting, methods
A total of 515 embryo videos and associated annotations were obtained from 502 patients. All videos were individually assessed and grouped according to the presence (n = 122) or absence (n = 393) of an excluded cell. Cell division annotations were collected from the 2-cell stage to blastocyst stage for each video. Time annotations and time intervals between subsequent cell divisions were analyzed to determine differences, if any, between control and exclusion embryos.

Main results and the role of chance
Following transfer, exclusion embryos resulted in significantly lower rates of fetal heartbeat (28% vs 48%; p &lt; 0.05) and live birth (20% vs 40%; p &lt; 0.05), compared to control embryos. A strong negative association was found between cell exclusion and reproductive success (OR = 0.41; 95% CI = 0.27-0.64; p &lt; 0.0001). Time annotations for 2-cell, 4-cell, and 6-cell to 9-cell stages in the exclusion group showed delayed cell divisions by an average of 3.16 hours up to morulation, compared to control embryos (p &lt; 0.05). However, times to 3-cell and 5-cell stages were significantly earlier than control embryos (p = 0.03 and p = 0.0002, respectively). Exclusion embryos seemed to spend more time in “intermediate” cell stages such as the 3-cell, 5-cell and 6-cell stages (p &lt; 0.01), compared to control embryos. Conversely, they spent less time in commonly known “checkpoint” stages such as the 2-cell, 4-cell, and 8-cell stage (p &lt; 0.001). Moreover, exclusion embryos were found to be more asynchronous than control embryos (p &lt; 0.0001) despite similar cell cycle lengths. No significant morphokinetic differences were found from the start of blastulation to full expansion before zona thinning, between experimental groups. Subgroup analysis revealed no significant trends for known confounding factors pertaining to cell division such as maternal age, paternal age, maternal body mass index and infertility diagnosis.

Limitations, reasons for caution
Aside from this study being retrospective, there may be unknown confounders that could impact this event. Moreover, identification of cell exclusion was conducted in a single data set generated by one clinic and was only performed by one person, thus limiting the generalizability of the findings.

Wider implications of the findings
Our results indicate that cell exclusion has negative associations with reproductive outcome. Moreover, morphokinetic analysis has been able to identify aspects of asynchronous and dysregulated cell division that are associated with cell exclusion. Further analysis combining morphokinetics with genetic testing and adhesion protein investigation may improve current embryo assessments.

Trial registration number
N/A