Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience

Abstract

Background: Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended.

Objectives: To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study).

Materials and methods: In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n = 17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n = 37), CFTR variants (n = 26), or variants in known infertility genes (n = 4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included.

Results: All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%).

Conclusion: ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

Keywords: asthenozoospermia; azoospermia; diagnostics; gene panel; genetic testing; genetics; male infertility; oligozoospermia; teratozoospermia.

FIGURE 1

A total of 130 genes were included in the first diagnostic panel version (DG‐3.6), with genes associated with azoospermia, oligozoospermia, asthenozoospermia, teratozoospermia, and fertilization failure. An overview of all genes and their associated phenotype is given in Table SIV. AZF, azoospermia factor; CBAVD, congenital bilateral absence of the vas deferens; MMAF, multiple morphological abnormalities of the sperm flagella; PCD, primary ciliary dyskinesia. Figure adapted from.

FIGURE 2

Schematic overview of the azoospermia factor (AZF) regions, sequence‐tagged sites (STS sites), and genes on the Y chromosome. The Y chromosome, and especially the AZFb and AZFc region, contains highly repetitive sequences. A graphic representation of the sequence organization is presented below the ideogram depicting the Y chromosome. Five palindromes (P1–P5) containing various amplicon sequence families (yellow, blue, turquoise, green, red, and gray) are depicted. For reference, transparent bands outline the amplicons in the background. The orange dots represent the STS sites used for detection of the classic AZFa (A), AZFb (P5/proximal P1; B) and AZFc (b2/b4; C) deletions according to the EAA/EMQN best practice guidelines (2014 and 2023)., In dark purple, the critical deletion region, and in lighter purple, the region in which the breakpoints are typically located. The large circles above the regions indicate the primary STS markers (two per region) used for deletion detection and the small circles indicate the STS markers used for the extension analysis. Below in red, the suggested genes for detection of deletion of the respective region. The other genes which can be used for extension analysis are depicted in green (single‐copy gene) and teal (multicopy genes). For an overview in tabular form, see Table SV.

FIGURE 3

Diagnostic results of exome sequencing (ES) in the implementation cohort. In A, the overall results of 292 infertile males are shown, with the clinically relevant variants divided into variants that could have been detected with karyotyping, AZF deletion screening, or CFTR analysis (CF‐EU2v1 kit, Elucigene Diagnostics), variants that are not detectable with those techniques, and carriership for cystic fibrosis (CF). These clinically relevant variants are further elucidated in B, while the phenotypes associated with the genes in which a monogenetic cause was identified with ES, other than CBAVD, are shown in C. AZF, azoospermia factor; CBAVD, congenital bilateral absence of the vas deferens; CNV, copy number variant; MMAF, multiple morphological abnormalities of the sperm flagella; SNV, single nucleotide variant.