P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis
Authors: Rodrigo L , Campos-Galindo I , Navarro R , Clemente M , Ferro A , Perez D , Akinwole A , Miravet J , Al Asmar N , Rubio C 
Published: July 2024

Study question

Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes?

Summary answer

PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need.

What is known already

Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative.

Study design, size, duration

In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study.

Participants/materials, setting, methods

The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol.

Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis.

Main results and the role of chance

The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo.

In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%).

Limitations, reasons for caution

The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases.

Wider implications of the findings

A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy.

Trial registration number

Not applicable