Targeted next-generation sequencing panel screening of 668 Chinese patients with non-obstructive azoospermia

Abstract

Purpose: We aimed (1) to determine the molecular diagnosis rate and the recurrent causative genes of patients with non-obstructive azoospermia (NOA) using targeted next-generation sequencing (NGS) panel screening and (2) to discuss whether these genes help in the prognosis for microsurgical testicular sperm extraction (micro-TESE).

Methods: We used NGS panels to screen 668 Chinese men with NOA. Micro-TESE outcomes for six patients with pathogenic mutations were followed up. Functional assays were performed for two NR5A1 variants identified: p.I224V and p.R281C.

Results: Targeted NGS panel sequencing could explain 4/189 (2.1% by panel 1) or 10/479 (2.1% by panel 2) of the patients with NOA after exclusion of karyotype abnormalities and Y chromosome microdeletions. Almost all mutations detected were newly described except for NR5A1 p.R281C and TEX11 p.M156V. Two missense NR5A1 mutations-p.R281C and p.I244V-were proved to be deleterious by in vitro functional assays. Mutations in TEX11, TEX14, and NR5A1 genes are recurrent causes of NOA, but each gene explains only a very small percentage (less than 4/668; 0.6%). Only the patient with NR5A1 mutations produced viable spermatozoa through micro-TESE, but other patients with TEX11 and TEX14 had poor micro-TESE prognoses.

Conclusions: A targeted NGS panel is a feasible diagnostic method for patients with NOA. Because each gene implicated explains only a small proportion of such cases, more genes should be included to further increase the diagnostic rate. Considering previous reports, we suggest that only a few genes that are directly linked to meiosis can indicate poor micro-TESE prognosis, such as TEX11, TEX14, and SYCE1.

Keywords: Male infertility; Microsurgical testicular sperm extraction; Next-generation sequencing; Non-obstructive azoospermia; Spermatogenesis.

Fig. 1

HE-stained sections of testicular tissue. Three figures demonstrate testicular phenotypes of varying severity in patients with NOA, including a normal spermatogenic function (A5197 with NR5A1 mutation), b maturation arrest (A961 with TEX11 mutation), and c Sertoli cell only (A2664 with KLHL10 mutation). Bar represents 75 μm. SC, Sertoli cells; SPC I and II, primary and secondary spermatocytes; SPT, spermatids

Fig. 2

In vitro functional assay of NR5A1 mutations. p.I224V and p.R281C were identified in this study. Analysis of three previously described variants p.G35E, p.R191C, and p.D238N were also repeated to increase credibility to our experiment. a Western blot analysis. b Assays of NR5A1 transcriptional activity by using downstream gene CYP11A1, CYP17A1, and CYP19A1. EV, empty vector; WT, wild type