Comprehensive genetic testing for female and male infertility using next-generation sequencing

Abstract

Purpose: To develop a comprehensive genetic test for female and male infertility in support of medical decisions during assisted reproductive technology (ART) protocols.

Methods: We developed a next-generation sequencing (NGS) gene panel consisting of 87 genes including promoters, 5' and 3' untranslated regions, exons, and selected introns. In addition, sex chromosome aneuploidies and Y chromosome microdeletions were analyzed concomitantly using the same panel.

Results: The NGS panel was analytically validated by retrospective analysis of 118 genomic DNA samples with known variants in loci representative of female and male infertility. Our results showed analytical accuracy of > 99%, with > 98% sensitivity for single-nucleotide variants (SNVs) and > 91% sensitivity for insertions/deletions (indels). Clinical sensitivity was assessed with samples containing variants representative of male and female infertility, and it was 100% for SNVs/indels, CFTR IVS8-5T variants, sex chromosome aneuploidies, and copy number variants (CNVs) and > 93% for Y chromosome microdeletions. Cost analysis shows potential savings when comparing this single NGS assay with the standard approach, which includes multiple assays.

Conclusions: A single, comprehensive, NGS panel can simplify the ordering process for healthcare providers, reduce turnaround time, and lower the overall cost of testing for genetic assessment of infertility in females and males, while maintaining accuracy.

Keywords: Clinical genetic testing; Diagnostic; Infertility; Next-generation sequencing.

Fig. 1

Description of the NGS panel by gene content, organized by the main infertility indications. Genes classified as “diagnostic” are shown in standard font, and genes classified as “informative” are shown in italic font

Fig. 2

a Outline of our NGS test. A DNA sample (saliva or blood) is sequenced by NGS and processed by our custom bioinformatics pipeline. Y chromosome microdeletions, sex chromosome aneuploidies, CFTR IVS8-5T polymorphism, indels, and SNVs are called. Variants are interpreted by expert curators and a medical report is generated. In parallel, FMR1 testing is performed using PCR and capillary electrophoresis, and results are incorporated into the medical report. b Example of Y chromosome microdeletion called in sample NA18337. c Example of IVS8-5T tract detection in sample NA19108, heterozygous for 5T/7T