This case-control study involved 60 non-obstructive azoospermic infertile men with isolated UDT and no gonadal dysgenesis, who were referred to the Royan Institute's infertility clinic between 2014 and 2019. The study was approved by the Ethical Review Board of the Royan Institute, following the principles outlined in the Helsinki Declaration (reference number: IR.ACECR.ROYAN.REC.1395.106). Blood samples were collected after obtaining informed written consent from all patients enrolled. The age of the participants ranged from 25 to 50 years at the time of referral. Seminal sample analysis was conducted after 3–5 days of sexual abstinence. Sperm concentration, motility, and morphology were assessed using the Sperm Class Analyzer system ver. 6.2.0.1 (Microptic Co.) following the World Health Organization (WHO) 2010 guidelines. The normal hormone ranges, as defined by the WHO, are 1.5–12.0 mlU/mL for FSH, 1–10 mlU/mL for LH, and 2–8 ng/mL for testosterone [16]. There is no definitive testosterone level below which a man can be conclusively diagnosed as hypogonadal. The Endocrine Society identifies 3 ng/mL (10.4 nmol/L) as a reasonable lower limit for normal total testosterone levels. The American Association of Clinical Endocrinologists recommends a threshold of 2 ng/mL [17]. Additionally, the International Society of Andrology, International Society for the Study of Ageing Male, European Association of Urology, European Academy of Andrology, and American Society of Andrology suggest that levels below 2.30 ng/mL typically indicate that patients might benefit from testosterone replacement therapy [18]. Therefore, an early morning total serum testosterone level below 3 ng/mL is a clear indicator of hypogonadism, while a level above 4 ng/mL in a healthy adult male patient generally rules out testosterone deficiency.

Hormonal levels in patients were measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit following the method described by Zangeneh et al. [19]. The inclusion criteria for the case group included isolated UDT, azoospermia, and normal karyotypes. Additionally, these patients exhibited several characteristics of hypergonadotropic hypogonadism, including elevated gonadotrophin hormone levels (FSH and LH), reduced testosterone levels, small testicular size, loss of facial hair, and decreased or absent libido. Normal adult testes are ovoid and measure approximately 3 cm (anterior-posterior)×2–4 cm (transverse)×3–5 cm (length), with a volume of 12.5–19 mL [16].

The exclusion criteria included microdeletions of the azoospermia factor (AZF) region, abnormal karyotypes [20], Wilms tumor, kidney diseases, and hypospadias. Cytogenetic analysis and AZF microdeletion detection were conducted using standard methods [21]. Additionally, all patients were screened for any history of genital tract infections known to cause male infertility, such as chlamydia, mycoplasma, and mumps. Those with a prior history of these infections were excluded from the study. Following these criteria, 60 men with non-obstructive azoospermia remained eligible for the study. They were categorized into groups with unilateral and bilateral UDT. The control group comprised 60 fertile men, each with at least one live-born child and no history of infertility or abortion. This group sought services at the Royan Institute for family balancing and sex selection. The ages of men in the control group ranged from 32 to 46 years old.

Total genomic DNA was extracted from 3 mL of whole blood using the salting-out method. For cell lysis, we utilized various buffers including red blood cell lysis buffer, cell lysis buffer, sodium dodecyl sulfate, and protein precipitation solutions. Additionally, RNase-A enzyme was added to eliminate any RNA present during the process. Subsequently, DNA precipitation was achieved by adding isopropanol and performing a wash with 70% ethanol. Finally, the DNA was solubilized and stored in Tris-ethylenediaminetetraacetic acid (EDTA) buffer. The DNA concentration was then measured using a Nanodrop Spectrophotometer 2000 (Thermo Fisher Scientific). The sequence of the WT1 gene was retrieved from the gene database available at https://www.ncbi.nlm.nih.gov/gene/7490. WT1 primers were designed using Perlprimer ver. 1.1.21 software (https://perlprimer.sourceforge.net) to amplify exons 2 and 9 of WT1 (Table 1). Polymerase chain reaction (PCR) was conducted using 50 ng of total DNA in a final volume of 25 μL, which included 10 μL of PCR Master Mix (Ampliqon) and 5 µM of each primer. PCR was performed on an Eppendorf PCR system with the following parameters: an initial denaturation at 94 °C for 4 minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, extension at 72 °C for 45 seconds, and a final extension at 72 °C for 8 minutes. After amplification, the PCR products were analyzed by electrophoresis on a 1.2% agarose gel and stained with GelRed (Biotium).

PCR products from gene amplifications were sent to Fazabiotech Company for DNA Sanger sequencing. The sequencing followed the Sanger method, utilizing an ABI 3730XL Capillary Sequencer (Applied Biosystems). The results were then compared with the normal sequence of the WT1 gene, using NC_000011.10 as the reference sequence from the National Center for Biotechnology Information (NCBI)-Gene database. Data analysis was conducted using Align Sequences Nucleotide Blast on the NCBI Blast server and Finch TV software ver. 1.4.0 (Geospiza Inc.).

In this study, the clinical characteristic data are presented as mean±standard deviation. Following the sequencing results, the data were processed and analyzed using SPSS software ver. 20 (IBM Corp.). The chi-square test was employed to analyze the genetic variables in the studied groups, with a significance threshold set at p<0.05.

Twenty-seven UDT cases were unilateral and 33 cases were bilateral during childhood. Most underwent orchiopexy surgery in the early years of their lives; however, the consequence of infertility became evident later in adulthood. Gonadotropin hormones (FSH, LH) and testosterone levels were evaluated in both the patient and control groups. The average hormone levels in patients and controls were assessed against the normal range, revealing characteristics of hypergonadotropic hypogonadism in the patients. Specifically, FSH and LH levels were significantly elevated in patients with infertile UDT, while testosterone levels were markedly lower than in the control subjects (Table 2).

Sequence analysis of the intronic boundaries of the second exon of the WT1 gene revealed a heterozygous intronic variation, rs34924443, in 34 patients (Table 3, Figure 1). This variation involves the insertion and/or deletion of G nucleotide(s) at position NC_000011.10:g.324287061. Although previously reported in the Single Nucleotide Polymorphism Database (dbSNP), this variation has not been documented in the Iranian population. It was also identified in 30 men within the control group. A comparison between the case and control groups showed no significant differences associated with this variation (p=0.4646). No specific transcription factors' binding sites were identified in this region. Additionally, while not reported in ClinVar (National Institutes of Health), this variant is listed as a common variant in the gnomAD browser database v3.1.2. Given its high population frequency, with an allele count of 36441 in 151034 (allele frequency: 0.2413), rs34924443 is classified as benign. Among these alleles, 3271 are from homozygous males from diverse ethnic backgrounds, including European, Ashkenazi Jewish, Latino, South Asian, African, East Asian, and other unspecified ethnicities. The age range of these individuals varies widely, from under 30 to over 80 years, and their parental status is not noted. Based on its high population frequency, rs34924443 is unlikely to contribute to male infertility and is considered a benign variant. Additionally, another intronic variant, rs573747031 (NC_000011.10: g.32428704G>T; NM_000378.6 (WT1): c.662-85C>T), was detected in the heterozygous form in one patient (Table 3, Figure 1). This variant is not listed in ClinVar. According to the gnomAD browser database v3.1.2, it has an allele count of 358 in 152138 (allele frequency: 0.002) and no instances of homozygous males have been reported. Despite its low allele frequency, in silico tools predict no impact on splicing or transcription factors' binding sites. Therefore, it is classified as likely benign according to the recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guideline.

After sequencing of exon 9, a heterozygous nucleotide variation, rs1851834762, was identified at the intronic boundary of this exon in a single infertile patient with UDT (Table 3, Figure 1). This novel intronic variation has not been observed in the Iranian population previously, nor was it detected in any individuals from the control group. Additionally, it has not been reported in ClinVar or gnomAD databases. This variation can be classified as likely benign since it does not affect splicing or transcription, and therefore has no impact on protein expression levels.

In addition, an exonic silent variant, rs374799820 (NC_000011.10: g.32392015G>A; NM_000378.6 [WT1]: c.1353C>T; NP_000369.4: p. Ser469=), was identified in the heterozygous state in an infertile patient with UDT (Table 3, Figure 1). This variant is listed in ClinVar (variation ID: 414085) and has been classified as likely benign according to ACMG/AMP guidelines.

A haplotype consisting of two heterozygous C>T variations was identified in the intronic region at the splice donor site of exon 9 in 11 patients from the case group, which included six with unilateral and five with bilateral UDT. These variations were absent in the control group. Statistical analysis revealed a significant difference between the case and control groups (p=0.022). One of these variations is already documented in dbSNP as rs587776576 (NC_000011.10: g.32391967C>T; NM_000378.4: c.1372+14G>A) and is depicted in Table 3, Figure 1. This variant, located at the splice donor site of +KTS/–KTS, is predicted to adversely affect a known splice site. Functional studies have confirmed its damaging impact and aberrant splicing, which disrupts the ratio of WT1 isoforms [22-25]. It is not present in the total gnomAD v3.1.2 dataset. ClinVar reports this variant (variation ID: 3493) and classifies it as pathogenic according to the ACMG/AMP guideline. Additionally, the allelic frequency of these alterations was found to be 9.16% in this study. Although the other novel intronic single nucleotide variation (NC_000011.10: g.32391969C>T; NM_000378.4: c.1372+12G>A), shown in Figure 1, appears to have no impact on transcription, splicing, or protein expression levels, it has not been observed in ClinVar or gnomAD. However, since it occurs as a haplotype with the known pathogenic variant rs587776576 and has a high calculated odds ratio of 28.11, its role in the pathogenicity of UDT or infertility in the Iranian population remains unclear.