Toward clinical exomes in diagnostics and management of male infertility

Abstract

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10^-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Keywords: monogenic infertility, azoospermia, oligozoospermia, cryptorchidism, exome sequencing, gene panel, molecular diagnostics, diagnostic yield, oligogenic, infertility management.

Graphical abstract

Figure 1

Likely pathogenic or pathogenic variants in 638 candidate genes identified in 521 men with spermatogenic failure and 323 normozoospermic men in the ESTAND cohort (A) Proportion of genes with findings in the analyzed panel. (B) Distribution of genes with LP/P variants according to the gene category. Fisher’s exact test, ^∗∗∗∗p ≤ 0.0001. (C) Singleton and digenic LP/P findings from the analyzed gene panel. (D) Cell-type-specific testicular expression (normalized log counts) of BNC1 from the human single-cell testis atlas (HISTA). (E) LP/P variants according to the previously reported inheritance mode. Fisher’s exact test, ^∗∗∗∗p ≤ 0.0001. (F) Stratification of LP/P variants by molecular consequence and novelty. (G) Genes with findings grouped by molecular consequence and novelty. (H) Distribution of unique and recurrent LP/P variants. (I) Number of LP/P variants identified per gene. (J) Genotype-phenotype data of the identified NR5A1 LP/P variants. AD, autosomal dominant; AR, autosomal recessive; BMI, body mass index; CR, cryptorchidism; ESTAND, ESTonian ANDrology; HPG, hypothalamic-pituitary-gonadal; LP, likely pathogenic; P, pathogenic; POI, primary ovarian insufficiency; sp, sperm; SPGF, spermatogenic failure; TV, testis volume; XL, X-linked; YL, Y-linked.

Figure 2

Examples of syndromic phenotypes and oligogenicity (A) Prediction of digenic pathogenicity using the ORVAL platform. Alternative colors denote individuals with two LP/P findings except for the blue stroked circles that report the analysis of three gene pairs from a man with trigenic findings (details in Table S11). (B) Pedigree with the segregating X-linked (XL) ATRX c.2542G>T (p.Asp848Tyr) variant. (C) Pedigree with the segregating autosomal-dominant (AD) variants PROK2 c.122G>A (p.Gly41Asp) and c.313C>T (p.His105Tyr) (from the gene panel), and KMT2D c.16051C>T (p.Arg5351Trp) (incidental finding). Variant validation by Sanger sequencing is provided in Figures S3 and S4. CR, cryptorchidism; d., died; DD, developmental disorder; FSH, follicle-stimulating hormone; hemi, hemizygous; het, heterozygous; LH, luteinizing hormone; T, testosterone; TTV, total testis volume; y, years.

Figure 3

Diagnostic yield and clinical characteristics of ESTAND infertile men with causal genetic findings (A) Diagnostic yield and affected gene categories in the clinical subgroups. (B) Affected genes stratified by primary diagnosis. (C) Comparison of the distribution of andrological parameters among men with genetic findings in each clinical subgroup (Mann-Whitney U test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). The box indicates the inter-quartile range with the median line, whiskers show the 1.5× inter-quartile range, and points show outliers. (D) Distribution of affected genes in men with Sertoli cell-only syndrome (SCOS). (E) Affected genes in SPGF-affected individuals reaching biological fatherhood. (F) The prevalence of low testosterone levels and cancer in SPGF subjects with genetic findings and the general male population (Fisher’s exact test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). (G) Other reported health conditions in SPGF-affected individuals with causal genetic findings. Detailed data are provided in Tables S10–S12. ART, assisted reproductive technologies; conc, concentration; CR, cryptorchidism; ej., ejaculate; ESTAND, ESTonian ANDrology; FSH, follicle-stimulating hormone; HPG, hypothalamic-pituitary-gonadal axis; LH, luteinizing hormone; NOA, non-obstructive azoospermia; OZ, oligozoospermia; POI, primary ovarian insufficiency; SPGF, spermatogenic failure; TV, testis volume.