PGT-A, a new dance for two couples: NGS with FISH and trophectoderm cells with blastocelic fluid

Abstract

Introduction Researchers are facing the problem of mosaicism detection and its reliability while conducting preimplantation genetic test for aneuploidy (PGT-A). Analysis of chromosome set in trophectoderm cells (TE) by the next generation sequencing (NGS) doesn't show the chromosome number in each cell of the embryo. Detection of certain types of mosaicism is also not available for NGS, but it can be revealed by fluorescent in situ hybridization (FISH). Blastocelic fluid (BF) contains DNA from both TE cells and inner cell mass,which can carry a different aneuploidy. Thus, obtaining the most accurate information on the embryo chromosome set is possible only by combining several study methods and material types. Therefore, the purpose of our study was to optimize the complex screening algorithm for embryo chromosome set analysis. Materials and Methods The study was performed in the Medical Centre IGR from March 2017 to February 2018. It involved 203 samples of embryos obtained from 55 women (mean age = 35.8±4.8 years) who were treated with IVF/ICSI cycles. TE biopsy and BF aspiration were performedon the post- fertilization hours 120 or 144. For a double diagnosis by NGS and FISH, a fragment of TE cells was divided into 2 pieces.107 samples were diagnosed using Ion S5 (Thermo Fisher Scientific, USA) and 96 samples - using FISH in chromosomes 9, 13, 15, 16, 17, 18, 21, 22, X, Y. The average number of nuclei from one sample analyzed by FISH was 3.4±1.3.Statistical analysis was carried out using Shapiro-Wilk test for normality, T-test for depend samples and Chi-square test. Results We analyzed the results of chromosome set study of TE cells by NGS verified by the FISH method. Among 67.1% (n=57) of samples with the same genetic characteristics by both methods, 44.7% (n=38) were euploid, 22.4% (n=19) - aneuploid. T-test for depend samples demonstrated no statistically significant difference (SSD) (p=0.32) in detection of sex chromosomes: 5.9% (n=5) of embryos had different combinations of sex chromosomes, e.g. XXY and XY, XXX and XX, XX and X0 by NGS and by FISH, respectively. Mosaicism study demonstrated SSD (p=0.0007) between NGS and FISH: 4.7% (n=4) and 18.8% (n=16), respectively. In all polyploidy cases detected by FISH (n=6), NGS demonstrated euploid chromosome set. Eight embryos (9.4%) detected as euploid by FISH were detected as aneuploid by NGS, because aneuploid chromosome wasn't included into the FISH panel.In 5.9% of embryos (n=5) aneuploidy was detected by both methods, but in different chromosomes. Among 11 embryos with BF and TE samples studied by NGS and FISH, 36.3% (n=4) showed euploid chromosome set without mosaicism, the same and different aneuploidies were detected in 54.6% embryos (27.3%, n=3, each), and in 1 embryo (9.1%) mosaicism was detected by FISH, but not by NGS, both in TE and BF. Conclusions The results show that the most expedient approach for the embryos selection in accordance with the results of PGT-A is to combine several methods and materials for the comprehensive study of embryo's genetic parameters.