Comprehensive Chromosome Screening (CCS) has flourished, progressing from early-stage Fluorescent In Situ Hybridization (FISH) to the current Array Comparative Genomic Hybridization (array CGH) technology for analyzing the entire genome. However, FISH has a poor resolution for chromosomes, while although array CGH can detect all 23 pairs of chromosomes, its ability to detect low-rate mosaicisms is limited in the context of pre-implantation genetic testing. With the advent of Next-Generation Sequencing (NGS), its advantages of speed, cost-effectiveness, and superior detection of mosaicisms have made it the mainstream technology in the market. Among the widely used sequencing methodologies are fluorescent markers and semiconductor sequencing technology.

Comparison of two platforms for PGT-A

This article focuses on the consistency of PGT-A results between the two platforms using automatic and manual identification. The following is a comparison of the throughput, hands-on time, turnaround time, and identification method between the two platforms.

Experimental Design and Procedures

The experimental design is divided into two stages, the first stage uses cell lines simulating different ratios of mosaic for sensitivity and specificity assessment, and the second stage is the clinical assay. The experimental workflow is shown in the figure below:

• After receipt of the sample, Lysis and Pre-Amplification will be performed first, and then the sample will be separated for follow-up experiments.

• In the automated identification platform, the DNA was then amplified and barcoded, and put into the Chef instrument with robotic arm for automated templating, then sequenced, and the experimental results were analyzed by the software.The table in the figure shows the results of the analysis, indicating the CNV of all chromosomes and their abnormal regions.

• In the manual identification platform, the library is prepared by hand, put into the instrument for sequencing, and then interpreted by human eyes.

The gray background is the common steps of both, the yellow background is the automatic identification platform, and the purple background is the manual identification platform. The thick box represents the automatic steps.

Result 1: Cell line sensitivity and specificity assessment

In the first stage, we simulated different ratios of mosaic DNA using cell lines and observed whether the Copy Number Variation (CNV) had the expected trend.

(figure 1-a) Automatic identification

(figure 1-b) Manual identification

Next, the sensitivity and specificity of the two platforms were evaluated using cell lines. Definition of sensitivity: ratio of abnormal detections to all abnormal detections (Figure 2-a); Definition of specificity: the ratio of detected normal detections to all normal detections (Figure 2-b).

The results showed that both platforms showed the expected trend. There is no statistical difference in sensitivity and specificity between the two platforms, whether automatic or manual.

Experimental result 2: Clinical examination consistency assessment

A total of 40 participants with a mean age of 37.2 ± 4.3 years were included in the second phase of the clinical trial. 589 eggs were retrieved, with 514 mature MII and 386 fertilized eggs (2PN). Finally, a total of 108 blastocysts were performed in PGT-A. One of them was not amplified successfully. First, we compared the chromosomal results of the blastocysts, and then analyzed the results of each chromosome pair in more detail, and the results are as follows:

The results showed 99.0% concordance for the blastocyst results and 99.5% concordance for each chromosome pair analyzed.

The consistency evaluation of the two platforms is summarized as follows:

1. Sensitivity and specificity assessments using cell lines are similar, with no statistical difference.

2. Whether analyzing blastocyst or chromosome pair results, the concordance rate is high.

3. In both PGT-A platforms, whether it is an automatic identification or manual identification, it has a high degree of accuracy and consistency.

The conclusion of this study is: no matter which PGT-A platform is used, the detection results with high consistency can be obtained. Advances in reproductive medicine allow us to use PGT-A plus correct implantation time, and exclude immune and thrombotic factors, the pregnancy rate can be as high as 80%!