Congenital absence of the vas deferens (CAVD) is found in 1%–2% of infertile men (Jequier et al., 1985). The clinical features of CAVD mainly present congenital bilateral absence of the vas deferens (CBAVD) or congenital unilateral absence of the vas deferens (CUAVD), the partial or total absence of the epididymal corpus and cauda, and absence or agenesis of seminal vesicles in the reproductive system (Weiske et al., 2000; Bieth et al., 2021). CAVD is generally identified during the evaluation of infertility due to azoospermia, known as one of the manifestations in cystic fibrosis (CF) (CF/CAVD) or accompanied by unilateral renal agenesis (URA) (URA/CAVD).

CF is a frequent disease in Euro-descendant populations, occurring in 1 of 2,000 newborns (de Souza et al., 2018). In Asian populations, the incidence varies greatly from 1:10,000 to 1:40,750 newborns among different countries (Powers et al., 1996; Yamashiro et al., 1997). Guo et al. reported that no more than 100 CF patients of Chinese origin were reported in the literature, which shows the incidence varies among different ethnic groups (Guo et al., 2018). A meta-analysis showed that the URA frequency in CUAVD patients was 26.8% compared with 6.7% in CBAVD patients (Cai et al., 2019).

The cystic fibrosis transmembrane conductance regulator (CFTR) was first identified by positional cloning in 1989, and its mutation was subsequently associated with CF and CAVD (Riordan et al., 1989; Dumur et al., 1990; Anguiano et al., 1992). Previous research demonstrated that CFTR homozygous or compound heterozygous variants (including the 5T allele) were as high as 78% in CBAVD patients, while only 46% in CUAVD patients (Yu et al., 2012; Cai et al., 2019). Almost all CF-related CBAVD patients had CFTR homozygous or compound heterozygous variants. X-linked ADGRG2 gene mutation has been verified to be associated with CAVD in recent years and is responsible for approximately 20% of CAVD not related to CFTR mutation (Patat et al., 2016; Bieth et al., 2021). However, CFTR or ADGRG2 or other mutations are rarely identified in CAVD men with URA, suggesting that other phenotypes with CAVD might have a different genetic background and need to be further researched.

Hypospadias is another common congenital malformation presented with hypoplasia of the penis. Compared with healthy children, boys born with hypospadias more often have other congenital anomalies. Hypospadias with CAVD is rarely reported in the literature. To our knowledge, hypospadias combined with the absence of the epididymis and vas deferens is only presented in complete androgen-insensitivity syndrome (CAIS) caused by androgen receptor mutation (Gottlieb et al., 1993). However, compared to CAVD patients, CAIS usually has impaired spermatogenesis. Hence, we assume that hypospadias with CAVD might have a genetic basis or pathogenic factor different from that of CF/CAVD and URA/CAVD.

The aim of this study was to evaluate the clinical features of the CAVD population, explore the mutation spectrum in CAVD patients, and further find potential causative genes by whole-exome sequencing (WES) in CAVD with hypospadias.

A total of 50 patients with CAVD were involved in this research, including 46 patients with CBAVD and four patients with CUAVD. Seven out of 50 CAVD patients had a history of hypospadias repair. Except for one patient who had chronic sinusitis, the other CAVD patients had no CF-related clinical features, such as progressive obstructive lung disease with bronchiectasis, pancreatic insufficiency, and others.

CFTR mutation is the most common genetic variation in CAVD populations, accounting for 60%–80% of CAVD patients (Chillon et al., 1995). In recent decades, with the advent of next-generation sequencing, several of these candidate genes such as ADGRG2 (Patat et al., 2016) and copy number variants (CNVs) in SLC9A3 (Wang et al., 2017) have been validated in some CAVD patients. Nevertheless, there is still no clear genetic diagnosis for approximately 20%–40% of CAVD patients. Hence, this study aimed to explore the variant spectrum in CAVD patients and to find new potential causative genes by WES in hypospadias/CAVD patients.

In this study, 29 out of 50 (58%) patients had at least one reportable CFTR variant. These results demonstrated that our CAVD population had a lower percentage than previous reports in which 70%–80% of CBAVD patients carried at least one CFTR mutation (Yu et al., 2012; Wang et al., 2020), which may be due to our small sample size. The variant c.1521_1523delCTT (F508del) is one of the most common variants in northern European CAVD populations, accounting for up to one-third of CAVD patients (Barratt et al., 2017; Bieth et al., 2021), whereas only two (2/50) are carriers in our CAVD patients. The 5T allele has a deleterious effect on the splicing of exon 10 and reduces the quantity of normal CFTR protein. The 5T allele polymorphism varies considerably by geographic location and ethnicity, particularly among non-Caucasian populations (Dork et al., 1997). In the current study, the frequency of the 5T allele was the most frequent variant, similar to other reports in Chinese cohorts (Du et al., 2014; Gaikwad et al., 2018; Wang et al., 2020). No ADGRG2 variants were identified, suggesting ADGRG2 variants may not play a major role in these CAVD populations. After Sanger sequencing, CFTR or ADGRG2 mutation was not detected in 21 CAVD patients, including seven hypospadias/CAVD patients. Therefore, WES was used to further test the potential pathogenic variants in hypospadias/CAVD patients. It is to be regretted that no homozygous or compound heterozygous mutations related to male infertility were identified.

In the hypospadias/CAVD group, the patients had a relatively lower testicular volume and higher FSH level than in the non-hypospadias/CAVD group, suggesting that their fertility, especially the capacity for spermiogenesis, maybe more deficient. As is known to all, cryptorchidism is the most common congenital anomaly associated with hypospadias (Leung and Robson 2007), but CAVD with hypospadias is rarely reported. CAVD is usually associated with some congenital diseases, such as CF-related CAVD and URA-related CAVD. Particularly, numerous studies have demonstrated that URA-related CAVD is less associated with CFTR mutation, suggesting the etiopathogenesis that may have a different genetic background.

No homozygous or compound heterozygous mutations related to disorders of sex development (DSDs) were identified by WES. Perhaps the hypospadias/CAVD population is so small that the CFTR mutation rate is not accurately evaluated or the two congenital abnormalities result from an embryonic development disorder occurring in the early stage of gestation. The embryonic anomaly may involve not only the formation of urethral folds but also the derivatives of Wolffian ducts. Nevertheless, nine heterozygous or hemizygous gene mutations were selected as possible pathogenic genes, such as SEMA3E, INVS, CBL, FRAS1, and AR. However, the phenotypic–genotypic linkage relationship has not yet been proven between these genes and hypospadias/CAVD patients.

The loss-of-function mutations in SEMA3E are formally associated with CHARGE syndrome, a complex multisystem genetic disease characterized by ocular coloboma, congenital heart defects, retardation of growth, genital hypoplasia, and facial asymmetry. (Song et al., 2020), but these hypospadias/CAVD patients had no other systemic abnormalities apart from genital hypoplasia. Mutation in INVS is usually involved in infantile nephronophthisis, but whether it leads to infertility has not yet been reported (Otto et al., 2003). A nonsense heterozygous mutation in CBL was identified in a patient. CBL which encodes an E3-ubiquitin ligase acts as a tumor suppressor in myeloid malignancies; also, its mutation is related to a Noonan syndrome-like disorder (Tartaglia et al., 2011). Noonan syndrome is a relatively common developmental disorder with a multisystem phenotype; however, our patients only have a genitourinary phenotype. The FRAS1 mutation is associated with Fraser syndrome. Although this syndrome presents genital anomalies, unilateral or bilateral cryptophthalmos is a typical clinical manifestation (Bouaoud et al., 2020). RSPO1 is one of the most important genes controlling female gonadal differentiation. Previous research has shown that RSPO1 mutation causes 46, XX testicular disorder of sex development, and that is contrary to relatively normal spermatogenesis in CAVD patients (Tallapaka et al., 2018). Mutations in the MKKS gene have also been shown to cause Bardet–Biedl syndrome which is characterized by pigmentary retinopathy, polydactyly, and renal abnormalities. Additionally, the MKKS null mouse model also failed to form spermatozoa flagella, which has no clear relevance in hypospadias or CAVD (Fath et al., 2005). A pathogenic variant in AR can cause androgen-insensitivity syndrome (AIS), which has undermasculinization of the external genitalia and impaired spermatogenesis, while hypospadias/CAVD patients have normal spermatogenesis. In the early stage of fetus development, testosterone promotes the formation of the internal reproductive structures (epididymis, vas deferens, prostate, and ejaculatory duct) from the Wolffian ducts, while dihydrotestosterone (DHT) induces the development of the external genitalia (Sultan et al., 2001). The level of testosterone in AIS is normal or increased, but it is normal or slightly lower in hypospadias CAVD patients. ATM encodes the ATM protein which activates cell-cycle checkpoints to repair the damaged DNA and prevent the presence of persistent deleterious lesions. In addition, ATM mutations may also be implicated in male infertility due to defective spermatogenesis (Li et al., 2013). Thus, the relevance of ATM to hypospadias CAVD needs to be verified more thoroughly.

Hypospadias is a common congenital condition that has been reported in over 200 syndromes, but only 30% of hypospadias cases have a clear genetic cause (van der Horst and de Wall 2017). Interestingly, few literature studies listed hypospadias in association with CAVD. All of the hypospadias/CAVD patients involved in our study were relatively healthy except for their infertility. Among the hypospadias/CAVD patients, the potential causative genes were not identified by WES in these patients, suggesting that further research needs to be carried out to elucidate the genetic background. Environmental factors, alteration of hormone levels, and epigenetic changes may play a role in the occurrence of hypospadias/CAVD.

In summary, CFTR mutations were screened in 50 CAVD patients to make a definitive genetic diagnosis and to evaluate their genetic risk. The 5T allele is the most common variant in our CAVD populations. In hypospadias/CAVD patients, neither CFTR mutations nor other homozygous or compound heterozygous variants related to disorders of sex development (DSDs) or male infertility were identified by WES, but it is necessary to carry out a more detailed exploration to determine the possible pathogenesis in hypospadias/CAVD.