Comprehensive Genetic Analysis of Non-Obstructive Male Infertility Using Combined Next-Generation Sequencing and Multiplex Ligation-Probe Amplification

Abstract Background Only a limited number of studies have investigated CFTR mutation status in non-obstructive infertile men. The aim of the present study was to evaluate the presence, frequency, and spectrum of CFTR mutations in infertile men diagnosed with non-obstructive oligozoospermia and azoospermia. Methods A total of 98 infertile men were included in this study. Based on semen analysis according to the 2021 World Health Organization (WHO) criteria, patients were classified into two groups: severe oligozoospermia and azoospermia. All patients had normal karyotype results and were negative for Y chromosome microdeletions. CFTR gene analysis was performed using both direct sequence analysis and multiplex ligation-dependent probe amplification (MLPA). Statistical analyses were carried out with SPSS version 22.0 (IBM Corp., Armonk, NY, USA). A p value < 0.05 was considered statistically significant. Results Among the study population, 31 patients (48.1%) had non-obstructive severe oligozoospermia and 67 patients (51.9%) had non-obstructive azoospermia. In total, 25 distinct CFTR mutations were identified in 27 patients (27.5%). The most frequent variant was IVS8-5 T (n = 3). Missense variants were the most common mutation type. Additionally, an exon 2 deletion was detected in one azoospermic patient by MLPA. No significant difference in CFTR mutation frequency or zygosity status (heterozygous vs. homozygous) was observed between the two groups. Conclusion Our findings suggest that all men with non-obstructive infertility should be screened for CFTR mutations. Comprehensive testing combining CFTR sequence analysis with MLPA provides a more accurate genetic evaluation compared to limited mutation screening.