ABSTRACT
OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.
Subject(s)
Blastocyst Inner Cell Mass/pathology , DNA Copy Number Variations , DNA Methylation , Epigenesis, Genetic , Epigenome , Epigenomics , Gene Dosage , Single-Cell Analysis , Trophoblasts/pathology , Aneuploidy , Blastocyst Inner Cell Mass/metabolism , Cell Separation , CpG Islands , Female , Gene Expression Regulation, Developmental , Humans , Trophoblasts/metabolism , Whole Genome Sequencing , WorkflowABSTRACT
PURPOSE: The aim of this study is to evaluate the correlation between serum estradiol (E2) levels during artificial autologous frozen embryo transfer (FET) cycles and ongoing pregnancy/live birth rates (OP/LB). METHODS: A historical cohort study was conducted in an academic setting in order to correlate peak and average estradiol levels with ongoing pregnancy/live birth rates for all autologous artificial frozen embryo transfer cycles performed from 1/2011 to 12/2014. RESULTS: Average and peak E2 levels from 110 autologous artificial FET cycles from 95 patients were analyzed. Average E2 levels were significantly lower in cycles resulting in OP/LB compared to those that did not (234.1 ± 16.6 pg/ml vs. 315 ± 24.8 pg/ml, respectively, p = 0.04). Although peak E2 levels were not significantly different between cycles resulting in OP/LB compared with those that did not (366.9 ± 27.7 pg/ml vs. 459.1 ± 32.3 pg/ml, respectively, p = 0.19), correlation analysis revealed a statistically significant (p = 0.02) downward trend in OP/LB rates with increasing peak E2 levels. CONCLUSIONS: This study suggests that elevated E2 levels in artificial autologous FET cycles are associated with lower OP/LB rates. Estradiol levels should be monitored during artificial FET cycles.
