This case–control study included 75 semen samples from males with abnormal results of the SDF test (cases) and 74 semen samples from males with normal SDF levels (controls). The study was ethically approved by the Ethical Research Committee at the Islamic University of Gaza (App. ID 28-2022); this study has also complied with the Declaration of Helsinki besides approved by the ethics committee, and all subjects gave their consent to participate in the study.

The reports of semen analysis, results of the SDF test, and general characteristics for all participants in this study were obtained from the records of Al-Basma IVF center in the Gaza Strip. Semen analysis included sperm count, liquefaction time, volume, morphology, motility, viscosity, and the percentage of abnormal sperm. The Spectrum Technologies (USA) HaloSperm kit was used to assess the level of DNA damage in semen samples. Three to four hundred sperm were examined, and an SDF of ≥ 30% was considered abnormal.

The genomic DNA was isolated from semen samples using the Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturerʼs instructions. The SNPs of the selected genes were genotyped using the allele-specific polymerase chain reaction (AS-PCR) technique. Specific PCR primers were designed using a web-based AS primer design application (http://bioinfo.biotec.or.th/WASP). Primer sequences are available from the corresponding author upon request. The PCR products were separated by running them on ethidium bromide-stained 3% agarose gels and visualized on a gel documentation system.

Data was collected, summarized, categorized, and analyzed using Statistical Package for Social Sciences (SPSS) software version 25. The frequencies of alleles and genotypes were compared between cases and controls by standard odds ratio (OR) at 95% confidence intervals (CIs) using an online calculator (https://www.medcalc.org/calc/odds_ratio.php). Hardy-Weinberg equilibrium (HWE) was tested using the online tool (https://wpcalc.com/en/equilibrium-hardy-weinberg/). All results were considered significant if the P-value was < 0.05. The multifactor dimensionality reduction (MDR) software (v.3.0.2) (http://www.epistasisblog.org/) was utilized to evaluate SNP-SNP interactions [5]. Interaction entropy graphs were created based on MDR results to identify synergistic and non-synergistic interactions among the variables.

As reported in our earlier work [6], the demographic characteristics and routine seminal parameters of the study population were as follows (Table 1): there were no statistically significant differences between the two groups in terms of mean age, BMI, smoking status, or IVF technique, as all P-values were greater than 0.05. The liquefaction time of the cases was significantly longer than that of the controls (P-value < 0.001), and the total motility of the sperm in the cases was significantly lower than that in the controls (P-value < 0.001). The sperm normal form in patients was also significantly different from that in controls (P-value = 0.049), with a lower proportion of patients having a normal form. However, there were no significant differences between cases and controls in terms of other seminal parameters, including viscosity, volume, and sperm count.

Analysis of seminal parameters revealed a statistically significant difference in liquefaction time between the groups (P-value < 0.05). This finding suggests that abnormal SDF may be associated with delayed or impaired semen liquefaction, affecting the release and motility of sperm. This result is consistent with a study conducted in Iraq [8].‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

The link between abnormal SDF and prolonged liquefaction time may be attributed to oxidative stress [9], inflammation, or other underlying factors associated with DNA damage. These factors can affect the composition and functionality of seminal fluid, ultimately resulting in impaired liquefaction.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

One of the studyʼs key findings was a significant decrease in total sperm motility among cases compared with controls (P-value < 0.001). Specifically, 56% of cases had sperm motility below 42%, compared with only 9.3% of controls. This significant difference highlights the impact of abnormal SDF on spermatozoaʼs overall motility. The observed decrease in total motility in cases compared with controls aligns with previous research that reported similar findings [10, 11].

The exact mechanisms connecting elevated SDF with decreased sperm motility are still not fully understood. However, it is possible that the DNA damage seen in sperm with abnormal fragmentation could disrupt the cellular processes necessary for efficient motility. Previous research has indicated that DNA damage in sperm could result in oxidative stress and mitochondrial dysfunction, both of which are associated with impaired sperm motility [12, 13].

In addition, the present study revealed a significant difference in the normal form of sperm between the cases and controls. This finding suggests that abnormal SDF may contribute to variations in sperm morphology. Previous research has established a link between abnormal sperm morphology and impaired sperm function, reducing fertility [14]. The association between abnormal SDF and altered sperm morphology can be attributed to several underlying mechanisms. DNA fragmentation in sperm may arise from diverse factors, including oxidative stress, exposure to environmental toxins, or genetic abnormalities. Additionally, sperm DNA damage has been associated with compromised chromatin compaction, which can influence the overall structure and morphology of the sperm [15].

Finally, there were no statistically significant differences between cases and controls regarding other seminal parameters, including viscosity, total semen volume, and sperm count. This result aligns with previous studies that reported no significant correlation between sperm count or semen viscosity and SDF [16, 17].

XRCC1, a crucial enzyme in the BER process, functions as a scaffolding protein that is essential for maintaining the stability of the BER pathway. Its main role is to recruit other necessary enzymes to the abasic site and coordinate their activities to enhance the efficiency of the pathway. The XRCC1 gene is highly expressed in the testes, particularly in pachytene spermatocytes and round spermatids. This consistent expression pattern is important for supporting spermatogenesis by aiding in DNA damage repair during meiosis and recombination in germ cells. As a result, mutations or variations in XRCC1 have the potential to disrupt the normal process of spermatogenesis, which is crucial for proper sperm production [18]. The rs25487 (p.Arg399Gln) polymorphism is a common genetic variation in the XRCC1 gene that has been extensively researched. This variation changes the amino acid from arginine, which is basic and positively charged, to glutamine, which is polar but uncharged at a conserved residue within the poly (ADP-ribose) polymerase binding domain of XRCC1 [19].

Genotypic analysis revealed that the TC genotype was significantly more prevalent in SDF-positive individuals (49%) than in controls (31%) (OR = 2.159; P-value = 0.024), suggesting an increased risk of SDF. Conversely, the CC genotype was significantly more frequent in the control group (66%) than in cases (43%) and was associated with a protective effect against SDF (OR = 0.379; P-value = 0.004). Although the TT genotype showed a higher prevalence in cases (8%) compared to controls (3%), this difference was not statistically significant (P-value = 0.171). These findings indicate a statistically significant association between the XRCC1 rs25487 polymorphism and susceptibility to SDF, particularly under codominant and dominant inheritance models. Our results are consistent with previous studies that reported associations between XRCC1 gene polymorphisms and idiopathic male infertility [18, 20, 21]. However, contrasting findings from other populations may be attributed to ethnic variability or differences in study design and sample size. Notably, the genotype distribution in the control group conformed to the HWE, suggesting the sample was genetically stable with respect to this SNP.

In spermatozoa, 8-oxoguanine DNA glycosylase 1 (OGG1) is an essential enzyme in the BER pathway. Both the sperm nucleus and mitochondria rely on this glycosylase, which actively removes 8-hydroxy-2ʼ-deoxyguanosine (8OHdG) and releases the adduct into the extracellular space [22]. Among many OGG1 variants, the most extensively studied one is rs1052133 C > G (p.Ser326Cys) [23]. This polymorphism has been linked to a decreased repair capacity [24].

In our study, there were no statistically significant differences in genotypic or allelic frequencies between SDF cases and controls (P-value > 0.05). However, both the CG and CC genotypes showed ORs below 1, suggesting a potential protective role. These findings align with those reported from Barcelona in 2018 [20]. A study from China revealed a significant association between GG and SDF [25]. The observed variations may be attributed to differences in sample sizes or ethnic diversity among the participants.

The distribution of OGG1 rs1052133 (C > G) genotypes in the control group deviated significantly from HWE, most likely due to the small sample size (n = 74).

The MUTYH gene encodes an adenine DNA glycosylase that removes mispaired adenines from the newly synthesized DNA strand when they are incorrectly paired with 8-oxoguanines.

The MUTYH gene variant rs3219489 G > C (p.Gln324His) results from an SNP causing an amino acid change from glutamine (Gln) to histidine (His) at position 324 of the MUTYH protein. This alteration in the nature of the two amino acids can impact the enzymeʼs function.

The CC genotype was more prevalent in SDF patients (31%) than in control men (18%), although the difference did not reach significance (P-value = 0.064). Similarly, the GG genotype did not significantly differ between the two groups, although it was less common in the control men (39% and 34%, respectively). However, the GC genotype significantly varied between the SDF patients and the controls (31% and 49%, respectively), with a P-value of 0.025. To date, no reports have investigated the potential impacts of the MUTYH Gln324His (G > C) SNP on seminal parameters or SDF. However, the results suggest that genetic SNPs can influence the efficiency of DNA repair processes, leading to the accumulation of DNA damage, potentially contributing to the development of various complex diseases and affecting semen quality and SDF.

In addition, a significantly increased risk of SDF with MUTYH, rs3219489 G > C, was observed under an overdominant genetic model (GG + CC versus GC) with a P-value = 0.02. Overdominant genetic inheritance implies that the heterozygous genotype confers a different phenotype than its homozygous counterpart does.

Furthermore, the genotype distribution in the control group adhered to HWE (P-value = 0.995), suggesting no major genotyping errors or population stratification.

APEX1 plays a crucial role in repairing apurinic/apyrimidinic sites that result from DNA cleavage by OGG1 and MUTYH while also aiding in the recruitment of other players in the BER process, such as DNA polymerase β and DNA ligase III [26]. Multiple genetic variations have been identified in this gene, including a G > T alteration in exon 5 that leads to the substitution of aspartic acid with glutamic acid (p.Asp148Glu; known as rs1130409) [23].

The distribution of APEX1 rs1130409 (G > T) genotypes in the control group was in HWE, as there was no significant deviation (P-value = 0.757) between the observed and expected genotypes. These findings suggest that the alleles for APEX1 rs1130409 (G > T) are randomly distributed in the study cohort.

The distribution of genotypes was as follows: GG genotype in 15 cases (20%) and 12 controls (16%); GT genotype in 23 cases (31%) and 34 controls (46%); and TT genotype in 37 cases (49%) and 28 controls (38%), with no significant differences (all P-values > 0.05). However, when the GG genotype was used as a reference, the OR for the GT genotype was 0.520 (P-value = 0.056), indicating a trend toward a decreased risk of SDF. These findings suggest that the APEX1 rs1130409 SNP may be associated with the risk of SDF, although the observed associations did not reach statistical significance. The trend toward a decreased risk of SDF in individuals with the GT genotype suggests that the heterozygous genotype may confer some protective effect against SDF.

To the best of our knowledge, there are no published studies on this polymorphism in relation to infertility or SDF. However, many studies have explored the associations between the rs1130409 SNP in APEX1 and various cancers [27–34].

The examined polymorphisms were not significantly associated with traditional semen parameters, such as motility, sperm count, or sperm morphology.

Importantly, the lack of statistical significance could be attributed to the relatively small sample size used in this study. A larger sample size would offer greater statistical power to identify significant associations. Additional factors, including environmental influences, lifestyle, and genetic variations, may play a role in the overall risk of SDF and should be taken into account in future studies.

The genotypic combinations with the highest risk for the four SNPs—MUTYH (G > C), XRCC1 (T > C), APEX1 (G > T), and OGG1 (C > G)—based on OR were (CC, CC, GT, CG) (OR = 7.193), followed by (CC, TC, GT, GG) (OR = 2.028), and finally (GG, CC, TT, CC) (OR = 1.690). However, these risk combinations did not show significant differences between the study groups.

The Fruchterman-Rheingold plot was utilized to assess the level of interaction among SNPs through entropy measurements [35]. As depicted in Figure 1, a synergistic interaction was observed between XRCC1 rs25487 and APEX1 rs1130409, potentially impacting an individualʼs vulnerability to DNA fragmentation in sperm. The figure also suggests a redundancy effect among the remaining SNPs.

This investigation involved Palestinian men residing in the Gaza Strip and experiencing increased SDF. The study focused on genetic variations in four SNPs: XRCC1 (p.Arg399Gln) rs25487, OGG1 (p.Ser326Cys) rs1052133, MUTYH (p.Gln324His) rs3219489, and APEX1 (p.Asp148Glu) rs1130409. The major outcomes of the study can be summarized as follows: elevated SDF is significantly correlated with low sperm motility, a low level of normal sperm form, and a high liquefaction time. Based on the frequencies of genotypes and alleles, the OGG1 C > G and APEX1 G > T polymorphisms are not linked to SDF in our study population. The MUTYH G > C polymorphism was associated with a lower risk for SDF, meaning individuals carrying the MUTYH (G/C) genotype may have a protective advantage against SDF. The XRCC1 rs25487 T > C polymorphism was associated with a greater risk for SDF in the study population with the (T/C) genotype. On the other hand, the genotype (C/C) is associated with a lower risk for SDF. The associations between XRCC1, rs25487, and SDF were significant under codominant, dominant, overdominant, and log-additive models, whereas the MUTYH rs3219489 polymorphism is significant under the overdominant model. There was no significant relationship between XRCC1 (T > C), OGG1 (C > G), MUTYH (G > C), or APEX1 (G > T) and the semen parameters (sperm count, motility, and sperm form) in our study sample. The highest SDF risk combination of the genotypes MUTYH (G > C), XRCC1 (T > C), APEX1 (G > T), and OGG1 (C > G) was CC, CC, GT, and CG, respectively.

The sample size in this study is relatively small, which limits the generalizability of the findings. Therefore, further validation using a larger and more diverse sample is necessary, particularly for the OGG1 C > G and APEX1 G > T gene polymorphisms.