A case–control study in Turkey has demonstrated that significantly elevated sperm DNA fragmentation and chromatin condensation abnormalities in oligozoospermic men occur independently of pathogenic mutations in the NBN gene. Published in the African Journal of Urology by Gülbahar Güzel Erdal and colleagues from Dicle University, the investigation evaluated fifty primary infertile men presenting with reduced sperm concentrations alongside fifty age-matched fertile controls from the same geographical region. Using targeted next-generation sequencing alongside quantitative cellular assays, the research team established that while men diagnosed with oligozoospermia exhibited substantial structural and nuclear defects in their spermatozoa, neither the patient cohort nor the control group harboured any pathogenic or likely pathogenic variants across the coding regions or splice junctions of the NBN gene. These findings indicate that increased sperm DNA damage in this cohort cannot be attributed to primary pathogenic mutations in this core DNA repair component, suggesting that other cellular pathways or environmental factors drive such reproductive impairments.

Infertility represents a major global health challenge defined as a couple’s inability to achieve conception following at least one full year of regular, unprotected sexual intercourse. Clinical evidence indicates that male-related reproductive factors account for approximately half of all diagnosed infertility cases, stemming from genetic anomalies, epigenetic dysregulation, physiological conditions, and environmental exposures. Recognized causes of male reproductive failure encompass hypogonadotropic hypogonadism, varicocele, cryptorchidism, Y chromosome microdeletions, testicular tumours, exposure to gonadotoxic medical therapies, and erectile dysfunction. In roughly fifteen percent of cases, infertility is classified as idiopathic because no underlying clinical etiology can be identified through standard diagnostic assessments. Within this landscape, sperm DNA fragmentation has emerged as a valuable biomarker of male reproductive potential, capturing structural damage that includes single-strand and double-strand breaks. Elevated fragmentation frequently arises from replication errors, excessive oxidative stress, apoptotic cascades, elevated scrotal temperatures, environmental toxins, lifestyle habits, or inadequate DNA repair capacity during spermatogenesis.

Among structural lesions affecting germ cells, double-strand breaks constitute an exceptionally grave threat to overall genomic stability. Failure or delayed resolution of these breaks during spermatogenesis is linked to chromosomal instability, permanent genetic alterations, reduced fertilization rates, impaired embryonic development, implantation failure, and an increased risk of miscarriage. Developing male germ cells rely primarily on two complementary mechanisms to repair double-strand breaks: homologous recombination and non-homologous end joining. Operating as an indispensable early sensor and coordinator of this damage response is the trimeric MRN complex, composed of MRE11, RAD50, and NBN. Upon recognizing structural lesions, this molecular assembly coordinates downstream biochemical signaling events by activating the ataxia telangiectasia mutated kinase, thereby regulating cell cycle checkpoints and orchestrating repair machinery. Within this complex, the NBN gene encodes nibrin, an essential scaffolding protein that maintains structural stability of the MRN assembly and facilitates critical signal transduction following genotoxic cellular damage.

The biological relevance of functional NBN is demonstrated by its link to Nijmegen breakage syndrome, a disorder characterized by chromosomal instability, defective cellular repair mechanisms, and impaired gametogenesis. Despite the established importance of the MRN complex in preserving genome integrity, the precise contribution of NBN variants to male infertility and sperm DNA integrity has remained uncertain. Earlier investigations into sequence variations in NBN and male reproductive outcomes yielded conflicting reports, with some literature suggesting potential associations with impaired spermatogenesis or abnormal semen parameters, while other studies observed no clear, reproducible, or independent effects. Prior studies had not established a consistent link between NBN gene alterations and sperm DNA fragmentation levels, prompting suggestions that NBN’s influence might be modest or context-dependent. Drawing on observations by Warcoin and colleagues regarding severe infertility phenotypes in biallelic pathogenic variant carriers, the research team designed this investigation to establish whether isolated oligozoospermia is driven by pathogenic NBN mutations.

The case group comprised fifty men presenting with primary infertility of at least one year’s duration and diagnosed with oligozoospermia, defined by World Health Organization 2021 standards as a sperm concentration below fifteen million cells per milliliter. The comparison group included fifty age-matched, demographically comparable normozoospermic men who had achieved spontaneous pregnancy with their partners within the preceding twelve months without a history of reproductive difficulties. Both study groups excluded individuals diagnosed with systemic chronic illnesses, grade three varicoceles, previous exposures to gonadotoxic chemotherapy or radiation treatments, recent histories of tobacco or alcohol consumption, or major surgical interventions within the preceding six months.

Semen sample procurement and laboratory processing followed standardized protocols, requiring two to three days of sexual abstinence before sample donation into sterile vessels. Semen specimens underwent initial macroscopic and microscopic evaluation based on World Health Organization standards before seminal plasma was removed via centrifugation at five hundred times gravity for seven minutes. Cellular pellets were washed with phosphate-buffered saline and preserved in a four percent formaldehyde fixative solution at four degrees Celsius. To measure sperm DNA fragmentation, the research team conducted terminal deoxynucleotidyl transferase dUTP nick end labeling assays using commercial diagnostic kits from Roche Diagnostics. Chemically fixed spermatozoa were spread onto polylysine-coated slides and permeabilized using a solution of zero point one percent sodium citrate and zero point one percent Triton X-100. Enzymatic staining proceeded at thirty-seven degrees Celsius for one hour, after which slides were washed in buffer and examined under fluorescence microscopy using DAPI and FITC filters, evaluating at least five hundred spermatozoa per sample with ImageJ to calculate the DNA fragmentation index.

In parallel with fragmentation assays, researchers conducted cytochemical analyses to quantify nuclear chromatin condensation defects via aniline blue staining. Sperm cells were rinsed twice in phosphate-buffered saline, concentrated through centrifugation at six hundred times gravity for ten minutes, and smeared onto glass slides. Smears were fixed in zero point seven five percent glutaraldehyde for thirty minutes, stained with five percent aniline blue in four percent acetic acid at pH two point five to three point zero for five minutes, and examined under oil immersion at one thousand times magnification. Additionally, while the oligozoospermic group underwent mandatory cytogenetic analysis via peripheral blood lymphocyte cultures and G-banding at a resolution of four hundred fifty to five hundred fifty bands per haploid set to exclude chromosomal abnormalities, fertile controls were qualified through proven recent fertility and urological examination.

Molecular genetic profiling focused on the coding sequence of the NBN gene using targeted next-generation sequencing. Genomic DNA was isolated from peripheral blood samples using QIAamp extraction kits on an automated QIAcube HT platform, followed by fluorometric quantification via Qubit dsDNA High-Sensitivity assays. Targeted sequencing libraries were prepared to evaluate all coding exons of the NBN gene, including exon–intron junctions spanning ten base pairs into flanking non-coding regions. The sequencing platform achieved an average read depth of approximately fifty-fold, providing a coverage rate of ninety-seven point fifty-six percent across the target regions. Detected variants were interpreted in line with formal American College of Medical Genetics and Genomics guidelines. Data distribution was tested using the Shapiro–Wilk test, and statistical comparisons were performed with independent-samples Student’s t-tests or Mann–Whitney U tests in SPSS version twenty-one, with significance established at p values below zero point zero five.

Comparative analyses demonstrated significant clinical divergence between cohorts despite matching demographic baselines. Oligozoospermic patients and fertile controls presented similar mean ages of thirty-two point four and thirty-one point nine years, respectively, alongside comparable body mass index values of twenty-five point seven and twenty-five point two kilograms per square meter. In contrast, semen volume, sperm concentration, total motility, and normal morphology rates were all significantly reduced in the oligozoospermic group, each yielding p values below or equal to zero point zero zero one. Functional cellular assays demonstrated marked differences: the DNA fragmentation index reached twenty-eight point seven plus or minus six point three percent in oligozoospermic men compared to eleven point two plus or minus three point one percent in fertile controls, reflecting statistical significance at p below zero point zero zero one. Aniline blue staining showed chromatin condensation defects affecting twenty-two point four plus or minus five point seven percent of oligozoospermic cells versus eight point seven plus or minus two point four percent in controls. However, targeted genetic sequencing identified no pathogenic or likely pathogenic NBN variants, detecting only common benign polymorphisms that showed no correlation with semen parameters or fragmentation rates.

The authors concluded that pathogenic NBN mutations do not represent a primary independent cause of elevated sperm DNA fragmentation in isolated oligozoospermia. While animal model studies establish that complete loss of NBN causes embryonic lethality, disruption of MRN complex components often impairs DNA repair processes without completely abolishing spermatogenesis, suggesting that compensatory mechanisms within interacting repair cascades may preserve basic germ cell development. The investigators emphasized several important study limitations, including the modest sample size of one hundred participants, which may have restricted detection of rare pathogenic variants. Furthermore, because sequencing utilized peripheral blood genomic DNA, testis-specific mosaicism, germ cell-restricted alterations, and localized epigenetic modifications could not be evaluated. Finally, genetic analysis was limited to the NBN gene, leaving other MRN complex members and ATM signaling components unexamined, indicating that sperm DNA fragmentation in this cohort reflects multifactorial repair pathways, epigenetic influences, and oxidative stress rather than isolated NBN gene mutations.

Subject of Research: Association between sperm DNA fragmentation and NBN gene variants in oligozoospermia

Article Title: Assessment of the association between sperm DNA fragmentation and pathogenic NBN gene variants in oligozoospermic men: a turkish case–control study

Article References: Güzel Erdal, G., Balkan, M., Tekeş, S., Oral, D., Güzel, R., Demir, Ğ., & Dede, O. (2026). Assessment of the association between sperm DNA fragmentation and pathogenic NBN gene variants in oligozoospermic men: a turkish case–control study. African Journal of Urology, 32(1), Article 45. https://doi.org/10.1186/s12301-026-00594-2

Image Credits: AI Generated

DOI: 10.1186/s12301-026-00594-2

Keywords: sperm DNA fragmentation, oligozoospermia, NBN gene, male infertility, MRN complex, TUNEL assay, next-generation sequencing, chromatin condensation, Assessment, association, between, sperm