Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes

Abstract

Study question: Could whole-exome sequencing (WES) be useful in clinical practice for men with maturation arrest (MA) after a first testicular sperm extraction (TESE)?

Summary answer: WES in combination with TESE yields substantial additional information and may potentially be added as a test to predict a negative outcome of a recurrent TESE in patients with MA.

What is known already: At present, the only definitive contraindications for TESE in men with non-obstructive azoospermia (NOA) are a 46,XX karyotype and microdeletions in the azoospermia factor a (AZFa) and/or AZFb regions. After a first negative TESE with MA, no test currently exists to predict a negative outcome of a recurrent TESE.

Study design, size, duration: In a cohort study, we retrospectively included 26 patients with idiopathic NOA caused by complete MA diagnosed after a first TESE.

Participants/materials, setting, methods: Twenty-six men with MA at the spermatocyte stage in all seminiferous tubules, according to a histopathological analysis performed independently by two expert histologists, and a normal karyotype (i.e. no AZF gene microdeletions on the Y chromosome) were included. Single-nucleotide polymorphism comparative genomic hybridization array and WES were carried out. The results were validated with Sanger sequencing. For all the variants thought to influence spermatogenesis, we used immunohistochemical techniques to analyse the level of the altered protein.

Main results and the role of chance: Deleterious homozygous variants were identified in all seven consanguineous patients and in three of the 19 non-consanguineous patients. Compound heterozygous variants were identified in another 5 of the 19 non-consanguineous patients. No recurrent variants were identified. We found new variants in genes known to be involved in azoospermia or MA [including testis expressed 11 (TEX11), meiotic double-stranded break formation protein 1 (MEI1), proteasome 26s subunit, ATPase 3 interacting protein (PSMC3IP), synaptonemal complex central element protein 1 (SYCE1) and Fanconi anaemia complementation group M (FANCM) and variants in genes not previously linked to human MA (including CCCTC-binding factor like (CTCFL), Mov10 like RISC complex RNA helicase 1 (MOV10L1), chromosome 11 open reading frame 80 (C11ORF80) and exonuclease 1 (EXO1)].

Large scale data: Data available on request.

Limitations, reasons for caution: More data are required before WES screening can be used to avoid recurrent TESE, although screening should be recommended for men with a consanguineous family background. WES is still a complex technology and can generate incidental findings.

Wider implications of the findings: Our results confirmed the genetic aetiology of MA in most patients: the proportion of individuals with at least one pathologic variant was 50% in the overall study population and 100% in the consanguineous patients. With the exception of MEI1 (compound heterozygous variants of which were identified in two cases), each variant corresponded to a specific gene-confirming the high degree of genetic heterogeneity in men with MA. Our results suggest that WES screening could help to avoid recurrent, futile TESE in men with MA in general and in consanguineous individuals in particular, but these results need to be confirmed in future studies before clinical implementation.

Study funding/competing interest(s): The study was funded by the Fondation Maladies Rares (Paris, France), Merck (Kenilworth, NJ, USA), IRSF (Montigny le Bretonneux, France) and Agence de la Biomédecine (Saint Denis, France). There are no competing interests.

Trial registration number: N/A.

Keywords: azoospermia; consanguinity; meiosis; non-obstructive azoospermia; spermatogenic arrest; testicular sperm extraction; whole-exome sequencing.

Figure 1.

Immunohistochemical analysis results for candidate variants in testicular seminiferous tubules of controls (X) and patients (X') with maturation arrest. A and A': CTCFL, CCCTC-binding factor like; B and B': SPATA22, spermatogenesis associated 22; C and C': SYCE1, synaptonemal complex central element protein 1; D and D': PSMC3IP, PSMC3 interacting protein; E and E': ZNF85, zinc finger protein 85; F and F': C11ORF80, chromosome 11 open reading frame 80; G and G': MAGEB6, MAGE family member B6; H and H': EXO1, exonuclease 1; I and I': CCDC36, coiled-coil domain-containing protein 36; J and J': FANCM, FA complementation group M; K and K': MOV10L1, mov10 like RISC complex RNA helicase 1; L and L': TEX11, testis expressed 11; M and M': MCMDC2, minichromosome maintenance domain containing 2; N and N': ARL2, ADP ribosylation factor like GTPase 2; O and O': MEI1, meiotic double-stranded break formation protein 1. Blue arrows = spermatogonia, black arrows = spermatocytes and orange arrows = round spermatids. Scale bars = 200 µm. ARL2, ADP ribosylation factor like GTPase 2; C11ORF80, chromosome 11 open reading frame 80; CCDC36, coiled-coil domain-containing protein 36; CTCFL, CCCTC-binding factor like; EXO1, exonuclease 1; FANCM, FA complementation group M; MAGEB6, MAGE family member B6; MCMDC2, minichromosome maintenance domain containing 2; MEI1, meiotic double-stranded break formation protein 1; MOV10L1, mov10 like RISC complex RNA helicase 1; PSMC3IP, PSMC3 interacting protein; SPATA22, spermatogenesis associated 22; SYCE1, synaptonemal complex central element protein 1; TEX11, testis expressed 11; ZNF85, zinc finger protein 85.

Figure 1.

Immunohistochemical analysis results for candidate variants in testicular seminiferous tubules of controls (X) and patients (X') with maturation arrest. A and A': CTCFL, CCCTC-binding factor like; B and B': SPATA22, spermatogenesis associated 22; C and C': SYCE1, synaptonemal complex central element protein 1; D and D': PSMC3IP, PSMC3 interacting protein; E and E': ZNF85, zinc finger protein 85; F and F': C11ORF80, chromosome 11 open reading frame 80; G and G': MAGEB6, MAGE family member B6; H and H': EXO1, exonuclease 1; I and I': CCDC36, coiled-coil domain-containing protein 36; J and J': FANCM, FA complementation group M; K and K': MOV10L1, mov10 like RISC complex RNA helicase 1; L and L': TEX11, testis expressed 11; M and M': MCMDC2, minichromosome maintenance domain containing 2; N and N': ARL2, ADP ribosylation factor like GTPase 2; O and O': MEI1, meiotic double-stranded break formation protein 1. Blue arrows = spermatogonia, black arrows = spermatocytes and orange arrows = round spermatids. Scale bars = 200 µm. ARL2, ADP ribosylation factor like GTPase 2; C11ORF80, chromosome 11 open reading frame 80; CCDC36, coiled-coil domain-containing protein 36; CTCFL, CCCTC-binding factor like; EXO1, exonuclease 1; FANCM, FA complementation group M; MAGEB6, MAGE family member B6; MCMDC2, minichromosome maintenance domain containing 2; MEI1, meiotic double-stranded break formation protein 1; MOV10L1, mov10 like RISC complex RNA helicase 1; PSMC3IP, PSMC3 interacting protein; SPATA22, spermatogenesis associated 22; SYCE1, synaptonemal complex central element protein 1; TEX11, testis expressed 11; ZNF85, zinc finger protein 85.

Figure 2.

Summary of results obtained in our case series, according to two different genetic strategies: target sequencing or whole-exome sequencing. CNV, copy number variations; SNV, single-nucleotide variant; TESE, testicular sperm extraction; TS, target sequencing; WES, whole-exome sequencing.

Figure 3.

A schematic representation of the genes involved in the major events in spermatogenesis. Candidate genes involved in maturation arrest (MA) in the present study or in other studies are shown in red and green, respectively. Other spermatogenesis-associated genes not yet described in human MA are presented in black. (A) The spermatogenesis process. Human MA genes can be involved in the proliferation of spermatogonial stem cells, the differentiation of spermatogonia, epigenetic modifications during meiosis, the protection of spermatocytes from retrotransposons and spermiogenesis. (B) Homologous recombination during prophase I comprises four substages: leptonema, zygonema, pachynema and diplonema. A series of events occurs throughout this period, including double-strand break (DSB) formation, 5′-to-3′ resection, strand invasion and crossover (double Holliday junction) formation and resolution. MA-causative genes can affect DSB formation and repair, and pairing, synapsis and recombination between homologous chromosomes. (C) Structure of the fusion between chromosome telomeres and the nuclear membrane during pachynema meiosis. During meiosis, telomeres attach to the inner nuclear membrane (INM) and drive the chromosome movement required for homolog pairing and recombination. Human MA-associated genes form complexes to accomplish this meiotic task. (D) The tripartite structure of the synaptonemal complex (SC), consisting of two parallel lateral elements and a central element. The SC normally forms between homologs during meiotic prophase I. Human MA-associated genes are involved in the formation of the SC complex and synapsis between homologous chromosomes.