Patients with azoospermia, aged 28 to 40 years, were chosen for the examination of promoter region polymorphism in the TNP2 and SYCP3 genes and microdeletions in AZFa. All patients received a thorough andrological examination, which included a medical history and physical examination, semen analysis, scrotal ultrasonography, and hormone analysis. In addition, every patient was examined to determine whether their karyotype was normal (46XY). A comprehensive medical history and physical examination were conducted for the controls, and all were fertile and had normal children.

In total, 100 men were selected for gene expression analysis as patient group after undergoing bilateral microdissection testicular sperm extraction (mTESE) to obtain spermatozoa for intracytoplasmic sperm injection. Karyotyping and Y chromosomal microdeletion analyses and the blood levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were assessed before surgery. The patients were not on hormone treatment and had primary infertility. Mean±standard deviation values for the serum LH, FSH, and testosterone levels of participants are illustrated in Table 1. None of the 100 male controls had a history of mTESE or cryptorchidism. The study excluded patients with cystic fibrosis, chromosomal abnormalities, and Y chromosome microdeletion. The control group consisted of 100 men with normal spermatogenesis (>15.0×10⁶ sperm/mL) according to the WHO criteria and confirmed fertility.

The research was approved by the local ethics committee (NO. 52/422370/1) and all subjects provided written informed consent. Blood samples from patients with azoospermia were collected to analyze gene polymorphisms and mutations. The salting-out DNA extraction technique was used to extract DNA samples from total blood samples.

To evaluate gene expression levels, frozen testis tissue was homogenized. Total RNA was extracted using the RNeasy Plus Universal Mini Kit (Qiagen) according to the manufacturer's protocol and stored at −80 °C. In-solution DNase digestion was used to eliminate DNA contamination. The concentration and purity of RNA were quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and confirmed by agarose gel electrophoresis. Template cDNA was synthesized from 1 μg of total extracted RNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) simultaneously with oligo-dT and random hexamer primers for each reaction in an Eppendorf Mastercycler Gradient (Eppendorf AG).

Polymerase chain reaction (PCR) amplification was performed using primer pairs (Table 2). PCR fragments were amplified as a 473bp fragment from the TNP2 gene, and the PCR conditions for TNP2 were as follows: 32 cycles of denaturation at 95 °C for 30 seconds, annealing at 57 °C for 1 minute, and extension at 72 °C for 30 seconds. The restriction fragment length polymorphism method was used for genotyping the TNP2 rs2857758 allele. The EarI restriction endonuclease enzyme was used to detect the CTCTTC sequence. The digestion reaction was carried out for 2 hours at 37 °C, and it digested 473-bp PCR products with altered G alleles into 353-bp and 120-bp fragments. The DNA fragments were isolated using 1.5% agarose gel electrophoresis and visualized in a gel documentation system by safe staining.

The presence of the T657C mutation in the SYCP3 gene was evaluated with the allele-specific (AS) PCR (ASPCR) technique. Two forward primers with variations in their 3' nucleotides were designed so that each was specific for one of the two variants (Table 2). They were combined with a common reverse primer into two PCR reactions. A 25-μL reaction mixture comprising 100 ng of DNA; 40 pmol of primers (SYCP3 F1, F2, and SYCP3 R); and 12.5 μL of 2x Taq PCR Master Mix was utilized for amplification. An initial melting phase of 94 °C for 5 minutes was followed by 35 cycles of 94 °C for 30 seconds, an annealing temperature starting at 60 °C for 30 seconds and 72 °C for 30 minutes for extension, and a final elongation step of 5 minutes at 72 °C. A gel documentation system was used to visualize the amplified DNA on 1.5% agarose gel with safe staining. The C and T alleles produced 286-bp bands due to the AS primers' reactions.

Multiplex PCR was employed according to the standard protocol for analysis of the AZF region of the Y chromosome. The AZFa subregion was analyzed using STS primers (Table 1). The European Academy of Andrology recommends these STS primers, which can identify 90% of the deletions in AZF loci (sY84, sY86 for the AZFa region). A total volume of 25L was used for the PCR amplification, including 100 to 200 ng of human genomic DNA as a template, 2.5 mM deoxynucleotide triphosphates (2.5 mM each of 2'-deoxythymidine-5'-triphosphate [dTTP], 2'-deoxycytosine-5'-triphosphate [dCTP], 2'-deoxyguanosine-5'-triphosphate [dGTP], 2'-deoxyadenosine-5'-triphosphate [dATP]), oligonucleotide primers (0.1 to 2.0 μmol/L each of the forward and reverse primers), 10x Taq DNA polymerase assay buffer (Tris with 15 mM MgCl₂), and 3 U of Taq DNA polymerase.

The conditions for thermocycling were standardized for the AZFa subregions, utilizing a TC-512 gradient thermocycler. PCR was performed on the samples in 35 cycles at 94 °C for 30 seconds, 53 °C for 45 seconds, and 72 °C for 60 seconds. Initial denaturation was performed at 94 °C for 5 minutes, followed by a final extension at 72 °C for 10 minutes. The PCR amplified products were stained with safe staining and identified on 2% agarose gel electrophoresis. Before loading the samples into the wells of the agarose gel, samples were mixed with gel loading dye in a ratio of 1:1.

Expression analysis was carried out by quantitative real-time PCR (qRT-PCR) on cDNA libraries using primers specific for TNP2, SYCP3, DDX37 (located within the AZFa region), and the housekeeping gene β-actin (Table 3). The expression of β-actin was examined as an internal control for RNA isolation and RT-PCR efficiency. Initial denaturation for RT-PCR started at 95 °C for 8 minutes, followed by 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. To avoid primer dimer amplification, the melting curve was created by raising the temperature from 72 to 95 °C. The qPCR was performed in triplicate on 48-well plates in the StepOne-Plus RT-PCR System (Thermo Fisher Scientific/Applied Biosystems) using 1.0 μL of produced cDNA, 10 μL of the SYBR Green qPCR Master Mix (Applied Biosystems ABI/PE), and 7.0 μL of DNase/RNase-free water. One microliter of designed primers was used for the gene expression profile. The average cycles to threshold (C_T) value was used for further analysis, and all RT-PCR cycles included non-template (cDNA) controls to rule out contamination. The comparative C_T method was used to analyze relative gene expression (2^-ΔΔCT). The 2^-ΔΔCT parameter displays the expression level (expressed as a fold change) with respect to the housekeeping gene.

The chi-square test was used to compare allele and genotype frequencies between case and control groups. Statistical tests of significance and chi-square analysis were performed using R programming version 4.0.5 (R Foundation for Statistical Computing). Differences in the allelic and genotypic frequencies of rs2857758 for TNP2 and rs769825641 for SYCP3 between case and control groups were evaluated using the chi-square test with an odds ratio to express the risk of single-nucleotide polymorphisms (SNPs) for the disease of interest. The Fisher exact test was used for statistical testing when the allelic count was less than 5.

The gene expression results were verified as the mean±standard error of mean. The Mann-Whitney U test was used for analysis of non-normally distributed data. Statistical analysis was implemented using Prism 6 (GraphPad Software) and p-values <0.05 were deemed statistically significant. Furthermore, receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were employed to assess the potential of these genes as diagnostic biomarkers.

The genotype frequencies for the rs2857758 SNP of the TNP2 gene on chromosome 16 (16p13.13) were 87% for T/T, 10% for T/G, and 3% for G/G in sterile males, whereas the corresponding frequencies among the control group were 76%, 15%, and 9%, respectively (Figure 1). The altered allele frequency was higher among normal controls than in the case group. However, no significant difference (p=0.053) was found between infertile and fertile males (Table 3).

SYCP3 rs769825641 polymorphism, located on 12q23.2, was genotyped using ASPCR. Two PCR reactions were performed, one with forward primer 1 for the T allele and the other with forward primer 2 for the C allele, run side by side on an agarose gel. A 286-bp band indicated the presence of the specific allele. We observed that 167 (83.5%) cases and controls were homozygous TT, 27 (13.5%) were heterozygous TC, and six (3%) were homozygous CC. The genotyping frequency difference between the case and control groups was statistically significant (p=0.04). Additionally, the frequency of mutant alleles was higher in the sterile men than in the fertile male volunteers (Table 4, Figure 2).

To validate the deletions in the AZFa region, the PCR reactions were repeated three times. The number of samples with deletions and their proportions are presented in Table 5. Deletions in the AZFa region were not found in patients with severe oligozoospermia. Among the 100 samples tested for the sY84 marker, four exhibited deletions in the AZFa region, and four showed deletions for the sY86 marker.

The cDNA libraries from testis tissue were screened by PCR for the TNP2, SYCP3, and DDX3Y genes with their specific primers. Based on the Mann-Whitney U test, SYCP3 and DDX3Y showed altered expression in the testis tissue of infertile patients compared to fertile male controls. In contrast, no significant alteration was observed for TNP2 expression among the infertile males as compared to controls (Figure 3).

ROC curve analyses were carried out to assess the diagnostic value of the TNP2, SYCP3, and DDX3Y genes for azoospermia. The results showed that SYCP3 and DDX3Y were potential biomarkers for azoospermia in fertile males, with AUCs of 0.772 and 0.720, respectively. However, TNP2 (AUC=0.568) showed no biomarker potential for diagnosing azoospermia (Figure 4).

The analysis of hormone levels indicated that both LH and FSH levels were significantly lower in infertile patients than in healthy controls (p<0.001). In addition, testosterone levels were significantly lower (p<0.001) (Figure 5).