Technique to ‘Map' Chromosomal Mosaicism at the Blastocyst Stage

Abstract

The purpose of this study was to identify a technique that allows for comprehensive chromosome screening (CCS) of individual cells within human blastocysts along with the approximation of their location in the trophectoderm relative to the inner cell mass (ICM). This proof-of-concept study will allow for a greater understanding of chromosomal mosaicism at the blastocyst stage and the mechanisms by which mosaicism arises. One blastocyst was held by a holding pipette and the ICM was removed. While still being held, the blastocyst was further biopsied into quadrants. To separate the individual cells from the biopsied sections, the sections were placed in calcium/magnesium-free medium with serum for 20 min. A holding pipette was used to aspirate the sections until individual cells were isolated. Individual cells from each section were placed into PCR tubes and prepped for aCGH. A total of 18 cells were used for analysis, of which 15 (83.3%) amplified and provided a result and 3 (16.7%) did not. Fifteen cells were isolated from the trophectoderm; 13 (86.7%) provided an aCGH result, while 2 (13.3%) did not amplify. Twelve cells were euploid (46,XY), while 1 was complex abnormal (44,XY), presenting with monosomy 7, 10, 11, 13, and 19, and trisomy 14, 15, and 21. A total of 3 cells were isolated from the ICM; 2 were euploid (46,XY) and 1 did not amplify. Here, we expand on a previously published technique which disassociates biopsied sections of the blastocyst into individual cells. Since the blastocyst sections were biopsied in regard to the position of the ICM, it was possible to reconstruct a virtual image of the blastocyst while presenting each cell's individual CCS results.

Chromosomal analysis in IVF: just how useful is it?

“…Capalbo et al (2013a) using an efficient immunostaining method to better characterize ICM and TE reported the rate of mosaic diploid-aneuploidy as 2.9% with no preferential allocation of abnormal cells between ICM and TE. To the best of our knowledge, however, there is only one small 'proof of principle' study (Taylor et al 2016) in which blastocyst was separated first into ICM and 4 TE quadrants and then further disaggregated into individual cells.…”

“…www.reproduction-online.org questions (Ottolini et al 2015). One glaring omission from the scientific literature at the moment, however, is a comprehensive cell-by-cell comparison of a large cohort of blastocyst embryos to establish overall levels of mosaicism in different germ layers (Taylor et al 2016). In fact, some of this may be estimated from preexisting NGS data; however, the question of level of meiotic vs postzygotic errors would benefit from whole embryo analysis.…”

Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst

“…ICM biopsies were isolated from vitrified-warmed blastocysts as outlined in the legend for Fig. 1A, basing the technique on a protocol described previously (Taylor et al, 2016) but omitting the exposure of samples to Ca 2+ /Mg 2+ -free medium. Immediately following ICM biopsy an additional TE biopsy was collected.…”

“…We adopted a modified ICM-biopsy procedure previously outlined (Taylor et al, 2016), which permitted us to collect an ICM biopsy and subsequently a second TE biopsy in blastocysts ( Fig. 1A and Video 1).…”

Mosaicism in preimplantation human embryos

“…Embryos with detected single or multiple chromosomal mosaicism in TE cells were thawed and incubated until complete expansion. Such blastocysts were sectioned in three parts with two containing TE cells and one -predominantly with ICM cells as previously described (Taylor et al 2016). WGA, sequencing, and qualitative DNA evaluation were performed for each sample as indicated above.…”