Extended In Vitro Culture of Human Embryos Demonstrates the Complex Nature of Diagnosing Chromosomal Mosaicism From a Single Trophectoderm Biopsy

摘要

The development of preimplantation genetic testing (PGT-A) in conjunction with assisted reproductive technology has led to valuable analysis of the high frequency of chromosomal abnormalities observed in human embryos. While mosaic embryos may lead to healthy live births, the likelihood of poor clinical outcomes is much greater when compared with euploid blastocyst development. Data regarding mosaic blastocyst developmental capacity are mostly derived from analysis of clinical outcomes, and there exists a need for blinded, nonselection studies to properly analyze in vitro fertilization outcomes following mosaic embryo transplantation. Mosaic embryos are typically prioritized for transfer based on specific abnormality and degree of mosaicism. Newly developed embryo culture technology allows blastocyst attachment, outgrowth formation, and extended culture for up to 14 days in vitro. This study leveraged an extended in vitro embryo culture system to analyze the effects of chromosomal aberrations and blastocyst mosaicism on early peri-implantation up to 12 days postfertilization (dpf). This study obtained data on blastocysts donated following PGT-A as well as standard in vitro fertihzation/intracytoplasmic sperm injection cycles. Next-generation sequencing (NGS) was validated for detection of mosaicism present in as low as 3 of 10 cells at a resolution of greater than 10 Mb. All NGS data analysis was done using the QDNAseq algorithm. Blastocyst quality was measured using the Gardner and Schoolcraft grading criteria, while outgrowth viability was assessed using morphological analysis. Fisher exact test (2-sided) was used to evaluate the association between blastocyst quality and blastocyst chromosomal profiles with culture outcomes. The study cohort included 80 good-quality blastocysts plated and cultures until 8 or 12 dpf. Of this cohort, 54 were PGT-A blastocysts, donated following an abnormal (n = 37) or mosaic (n=17) diagnosis. All of these blastocysts formed outgrowths at 8 dpf. Of the 73 embryos cultured until 12 dpf, 51% remained attached, showed good developmental progression, and were determined to be viable. A total of 36 embryos in this group originally presented with 1 (n = 24) or multiple (n=12) aberrations, whereas 18 were reported as mosaic. The majority of these mosaic embryos had a single mosaic abnormality (n = 10), with the remaining having multiple (n = 2) or both uniform (complete) and mosaic abnormalities (n = 6). Euploid blastocysts and those diagnosed with trisomies, duplications or mosaic aberrations had the highest rate of viable 12 dpf outgrowths (34 of 44 attached [77%]). Monosomies, deletions, and chromosomal constitutions consisting of multiple aberrations significantly impaired in vitro embryo development to 12 dpf (3 of 29 attached [10%]; P < 0.0001). All embryos diagnosed with multiple aberrations were nonviable at 12 dpf. Notably, a significant number of embryos originally diagnosed as mosaic remained viable at 12 dpf (58%). Sensitivity and specificity were examined for detecting mosaicism using the study team's approach, resulting in an 18.5% false-positive rate and 0% false-negative rate for accurate diagnosis of euploid for all chromosomes expected to be normal. When structural and mosaic abnormalities are removed from this analysis, diagnostic accuracy increases from 80% in the context of PGT-A to 100%, with a 0% false-positive and false-negative rate.