NGS-Based Application for Routine Non-Invasive Pre-Implantation Genetic Assessment in IVF

Abstract

Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF centre have not been started in the absence of a recommendation. Our objective in this study was to provide a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology with the corresponding bioinformatic pipeline. In a retrospective study, we performed NGS on spent blastocyst culture media of Day 3 embryos fertilised with intracytoplasmic sperm injection (ICSI) with quality score on morphology assessment using the blank culture media as background control. Chromosomal abnormalities were identified by an optimised bioinformatics pipeline applying copy number variation (CNV) detecting algorithm. In this study, we demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A that can be carried out within 48 h, which is critical for the same-cycle blastocyst transfer. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.

Keywords: bioinformatic pipeline; in vitro fertilisation; missed abortion; next-generation sequencing; screening; spent culture medium.

Figure 1

Representing plots from the raw data quality checking process with the following subfigures: (a) Sequence quality histogram, (b) Sequence duplication level, (c) Per sequence GC content, (d) Adapter content.

Figure 2

Representing plots from the analysis of mapping quality metrics of the selected samples. The subfigures represent the following results: (a) Coverage histogram showing the genomics bin counts with the corresponding coverage, (b) Cumulative coverage genome fractions showing the fraction (%) of the genome which has at least “X” coverage, (c) GC content distribution of the mapped reads where the dashed lines corresponds to the theoretical distribution.

Figure 3

Odds ratio analysis for CNV in culture media droplets of aborted embryos (Missed) compared to control media and culture media droplets of healthy neonates (Healthy) compared to control media. Odds ratios are in log transformed scale for better visualization.

Figure 4

Karyogram representing clinically relevant autosomal alterations identified based on the NGS analysis of the gDNA content from the culture media of the aborted embryos. Dark red bands showing the centromeres, green bands above the chromosomes are indication gains and dark blue bands showing losses.

Figure 5

Representation of the entire workflow with all four main steps including Step 1: IVF procedure and sample collection, Step 2: Whole genome amplification. Step 3: Next-generation sequencing and Step 4: Bioinformatics analysis.