The genetic diagnosis of male infertility represents a very important step, but at the same time, it is a difficult challenge. On the one hand, spermatogenesis is a complex mechanism, and various conditions can compromise it; on the other hand, more than 2000 genes are involved in this multifaceted process. Therefore, a genetic diagnosis can remain elusive in many cases . Nowadays, NGS, a widely used method, allows the evaluation of a large number of genes simultaneously, rapidly, and inexpensively. However, the large number of gene variants that can be identified is virtually unlimited and may exceed the ability to validate them with functional studies. Therefore, bioinformatics is set to become an increasingly important tool.
Few studies have evaluated mutations in genes known to cause SPGF in NOA patients with known testicular histology. A recent study by Krausz and colleagues assessed a panel of genes involved in meiosis in 147 patients with NOA and maturation arrest. At least 12 genes (ADAD2, TERB1, SHOC1, MSH4, RAD21L1, TEX11, TEX14, STAG3, MEIOB, DMRT1, MEI1, and SYCE) have been found associated with maturation arrest . Therefore, the authors suggested that these genes may represent a gene panel to be used before sperm retrieval by testicular sperm extraction (TESE) in NOA patients. However, mutations/variations of the same gene seem capable of causing different testicular histological pictures. For example, partial deletion of the TEX11 gene resulted in maturation arrest in the study by Krausz and colleagues , but other mutations (e.g. splice region and intron or missense) of the same gene are compatible with different testicular histological findings such as hypospermatogenesis (case ID 125687856 of the present study) or with oligozoospermia .
The present study was designed to understand whether variations of a homemade panel of genes involved in spermatogenesis can be associated with specific testicular histology in patients with NOA. The panel was developed based on our previous review study . Only SPGF-associated penetrating genes were selected. Furthermore, only those with more significant negative consequences (splicing, frameshift, missense, start loss, deletions) were chosen to reduce the possibility of benign variants. In contrast, those with a frequency greater than 0.05 in the general population were excluded. We found pathogenic variants of the following five genes in six patients (12.5%) using this panel.
DNAH1 encodes for a protein that belongs to the dynein family of proteins. These are microtubule-associated motor protein complexes. Specifically, the DNAH1 protein, an inner arm heavy chain dynein, has been identified in the full length of the sperm flagellum, and the gene mutations have been classically associated with multiple morphological abnormalities of sperm flagella . However, DNAH1 mutations have been recently suggested deserving investigation not only in patients with asthenozoospermia but also in those with azoospermia . Indeed, an observational study has analyzed DNAH1 gene mutations in a cohort of 200 patients with NOA, reporting the presence of pathogenic variants in 3 of them (1.5%) . Accordingly, mutation of the DNAH5 gene, encoding for another component of the dynein family of proteins, has already been reported in a patient with NOA [15, 16]. On this basis, the possible role of the DNAH1 gene in the pathogenesis of NOA needs to be further elucidated. We herein report a pathogenic c.6058G>T stopgain mutation of the DNAH1 gene in a patient with SCOS, borderline serum FSH levels, low testicular volume, and cryptorchidism. These data support the role of this gene in NOA.
NANOS1 encodes for a transcript involved in the modulation of germ cell proliferation and, therefore, acts in the very early phase of spermatogenesis . Few reports have already suggested the link between mutations of this gene and SPGF. The heterozygous deletion of two single amino acids (p.Ser78del or p.Ala173del) has been reported in Polish patients with NOA, low testicular volume (6-10 ml), and elevated serum FSH levels (15.4-18.2 IU/l). The testicular histology was available only in one patient and showed SCOS . In addition, the pArg246His and Arg276Tyr NANOS1 missense mutations have been described in patients with severe oligozoospermia . Here we show a pathogenic frameshift NANOS1 mutation in a patient with cryptorchidism, low testicular volume, borderline FSH values, and mixed atrophy. Our findings expand the knowledge on the testicular histological features that can associate with NANOS1 mutations.
PLCZ1 encodes a protein of 608 amino acids expressed in spermatozoa and, specifically, in the acrosomal and post-acrosomal regions, in the intermediate tract, and the main part of the tail . This protein represents the molecular trigger for the oocyte activation during fertilization, as human PLCZ1 has been observed to stimulate the activation of the mouse oocyte and the embryo's development up to the blastocyst stage . Furthermore, PLCZ1 has been identified as a marker of human sperm health, and this protein's total expression levels have been correlated with sperm parameters . We found the c.20del frameshift pathogenic mutation of the PLCZ1 gene in a patient with apparently idiopathic NOA. He has normal testicular volume and serum FSH levels and hypospermatogenesis. There are no other studies that relate PLCZ1 to patients with NOA, most probably because the mutation of this gene is not usually searched in these patients. Further studies are needed to clarify better whether mutations of this gene are associated with hypospermatogenesis.
ZPBP encodes for a protein of 351 amino acids involved in acrosome compaction. The absence of this protein leads, in mice, to the fragmentation of the acrosome and the disruption of the Sertoli-spermatid junctions. These structural changes result in dysmorphic spermatozoa with a decreased ability to penetrate the zona pellucida . However, disruption of the Sertoli-spermatid junction can also lead to abnormal spermatogenesis, resulting in azoospermia . Despite this, no studies have described ZPBP gene mutations in patients with NOA. For the first time, we report ZPBP gene mutations in two patients with NOA, one with hypospermatogenesis and one with maturation arrest/mixed atrophy. This finding suggests that ZPBP gene mutations may play a pathogenic role in patients with NOA.
ZMYND15 encodes for a protein expressed in the testis, containing a zinc finger MYND motif and a nuclear localization signal. It is predicted to be a transcriptional repressor controlling normal temporal expression of haploid cell genes during spermiogenesis . Mutations of this gene have already been associated with azoospermia due to maturation arrest  and in patients with severe oligozoospermia . Herein, we show the presence of a pathogenic mutation of this gene in a patient with hypospermatogenesis. Interestingly, the expression of ZMYND15 mRNA in the seminal plasma may predict successful sperm retrieval with high sensitivity and specificity. Indeed, its expression was significantly decreased in patients with NOA and no sperm retrieval compared with NOA and successful sperm retrieval .
We found several VUS of the SPGF genes evaluated in patients with NOA. The majority of these VUSs are compatible with their testicular histological features but cannot be described as pathogenic because of the lack of functional studies. At least some of them will likely be considered pathogenic, thus further raising the diagnostic rate found in the present study. Among the genes we tested, we would like to underline the high prevalence of VUSs of the USP9Y gene and, in particular, the c.7434+14del variant. Furthermore, very interestingly, the USP9Y VUSs were found associated with different testicular histological features. These include SCOS, mixed atrophy, and hypospermatogenesis regardless of the presence of cryptorchidism or varicocele since they were also found in patients with idiopathic NOA. These findings lead to at least two major considerations: 1) the USP9Y gene may play a role in the pathogenesis of NOA; 2) there is not a direct correlation between a gene and specific testicular histology. More likely, the mutation/variation of a specific gene involved in spermatogenesis may cause different testicular histological features. This concept challenges the construction of custom-made gene panels for TESE prognostic purposes.
The USP9Y gene encodes for the ubiquitin-specific protease 9 and maps within the AZFa region of the Y chromosome. The hypothesis suggesting the role of the USP9Y gene in spermatogenesis failed when a case report documented normal spermatogenesis in a man with the complete deletion of this gene . However, this is in sharp contrast with the mild testicular phenotype reported in two cases with complete USP9Y deletion . Hence, still today, the role of this gene in spermatogenesis is a matter of debate. The data of the present study may suggest an association between VUSs of this gene and NOA.
The results of the present study must be interpreted with caution because of the absence of a control group consisting of fertile men and the relatively low sample size. However, the stringent inclusion criteria, the exclusion of all possible acquired causes of NOA, and the availability of the testicular histology for each patient included in this study represent its main strengths.
In conclusion, the results of the present study showed that a relevant percentage of patients with apparently idiopathic NOA have mutations of SPGF genes. Furthermore, the data, taken together with those of other studies [6,8,9,17], suggest that not the gene itself but the type of mutation is associated with testicular histology. In fact, different mutations of the gene at stake cause various testicular histological features. This challenges the design of a gene panel with predictive value for sperm retrieval by TESE in patients with NOA.