Understanding the impact of lifestyle and demographic factors on DNA fragmentation index (DFI) among infertile men.

This is a retrospective cohort study that utilized data obtained from a study carried out between March 2021 and December 2023 at three health facilities of Nordica Fertility Centre in Lagos, Asaba and Abuja cities in Nigeria. This retrospective study included data from 643 male patients who underwent DFI analysis at three health facilities of Nordica Fertility Centre in Lagos, Asaba and Abuja cities in Nigeria. Data on demographic characteristics (age, occupation), lifestyle factors (BMI, alcohol use, smoking habits, frequency of ejaculation), medical history, PICSI binding results, sperm motility, and pregnancy outcomes were collected from patient records. Female partners included in the embryo transfer and pregnancy analysis were between 25 and 38 years old, had normal ovarian reserve (AMH > 1.0 ng/mL), and were free from uterine abnormalities. Exclusion criteria included prior poor ovarian response, endometriosis, or systemic conditions that may affect implantation. All participants underwent controlled ovarian stimulation using standard antagonist protocols, followed by blastocyst-stage embryo transfer. Pregnancy was confirmed by a serum β-hCG level > 25 IU/L 14 days post embryo transfer and was further validated via ultrasound at 6–7 weeks for the presence of a gestational sac. The pregnancy rate was calculated per embryo transfer cycle. Only cycles with complete follow-up were included in this analysis. Based on sperm DFI levels, the patients were categorized into two groups: (1) those with a DFI ≤ 25% and (2) those with a DFI > 25% [ 19 ]. Potential risk factors, such as alcohol consumption, smoking, BMI, and age were then analyzed within each group. Men presenting for infertility evaluation at Nordica Fertility Centre were recruited for this study. The inclusion criteria were: Males aged 20 years and above. at least 12 months of unprotected intercourse without conception and. availability of complete semen analysis results, including DNA fragmentation index (DFI) assessment. Males aged 20 years and above. at least 12 months of unprotected intercourse without conception and. availability of complete semen analysis results, including DNA fragmentation index (DFI) assessment. while the exclusion criteria included men with: A history of recent febrile illness, specifically within three months. known genetic disorders affecting sperm function. history of chemotherapy or radiotherapy and. recent use of drugs known to affect sperm parameters. A history of recent febrile illness, specifically within three months. known genetic disorders affecting sperm function. history of chemotherapy or radiotherapy and. recent use of drugs known to affect sperm parameters. Semen samples were collected by masturbation after 2–7 days of sexual abstinence into sterile containers and allowed to liquefy at room temperature (22–27 °C) for 30 min. Semen analysis was conducted according to the World Health Organization (WHO) 6th edition laboratory manual for the examination and processing of human semen. The parameters assessed included: Sperm Concentration (10⁶/ml): This was determined using a Neubauer counting chamber after proper dilution. Motility (%): This was categorized and expressed as a percentage of total sperm. Morphology (% normal forms): This was assessed using strict Tygerberg criteria with Papanicolaou-stained slides. DFI (%): This was measured using the Halosperm ® kit (Halotech DNA, Spain). Sperm Concentration (10⁶/ml): This was determined using a Neubauer counting chamber after proper dilution. Motility (%): This was categorized and expressed as a percentage of total sperm. Morphology (% normal forms): This was assessed using strict Tygerberg criteria with Papanicolaou-stained slides. DFI (%): This was measured using the Halosperm ® kit (Halotech DNA, Spain). To improve clarity and precision, motility was reported as actual percentages. Participants’ semen parameters were stratified by age groups ( 45 years) to assess age-related differences. The sperm DNA fragmentation index (DFI) was measured using the Halosperm ® kit (Halotech DNA, Spain), a commercial version of the Sperm Chromatin Dispersion (SCD) test, as described by Fernández et al. [ 20 ]. This assay is based on the principle that sperm with intact DNA form large or moderate halos of dispersed chromatin following acid denaturation, whereas fragmented DNA results in small or no halos. Briefly, semen samples were diluted to 10 million sperm/mL in phosphate-buffered saline (PBS) and mixed with liquefied low-melting-point agarose. The mixture was placed on pre-coated slides, allowed to solidify at 4 °C, and then subjected to a standardized series of treatments: Acid denaturation for 7 min. Lysis for 25 min. Washing and ethanol dehydration (70%, 90%, 100%). Staining with Wright’s solution for bright-field microscopy or DAPI for fluorescence microscopy. Acid denaturation for 7 min. Lysis for 25 min. Washing and ethanol dehydration (70%, 90%, 100%). Staining with Wright’s solution for bright-field microscopy or DAPI for fluorescence microscopy. Two slides per patient were analyzed. On each slide, 1,000 spermatozoa were evaluated under bright-field microscopy. Sperm DNA fragmentation was determined as the proportion of sperm displaying small or no halos. Based on standard interpretation guidelines, results were categorized into four groups:  50%– High fragmentation (low fertility potential).  50%– High fragmentation (low fertility potential). For the purpose of this study, DFI ≤ 25% was classified as good sperm DNA integrity, while DFI > 25% was classified as poor sperm DNA integrity. The data collected were analyzed using STATA version 16.0 for Windows (Stata Corps, College Station, Texas 77845, USA). The chart was done using Python 3.11. Descriptive statistics were used to summarize the demographic and lifestyle characteristics of the study population. Chi-square tests were used for categorical variables to assess initial associations between DFI levels and categorical variables. Sperm DNA fragmentation index (DFI) was categorized using a 25% threshold, with DFI ≤ 25% classified as good sperm DNA integrity and DFI > 25% as poor integrity. This threshold serves as the basis for the binary classification in the logistic regression analysis based on the assay used. The logistic regression model was used to control for potential confounders and determine independent predictors of high DFI (> 25%). For the logistic regression model, DFI was the dependent variable. Independent variables included age (recoded as < 45 vs. ≥45 years), occupation, frequency of ejaculation, presence of medical conditions, sperm motility category, number of embryos transferred, PICSI-binding rate, alcohol consumption, and smoking status. The categorization of occupations in this study as Professional/Technical/Managerial, Clerical, Sales and Services, Skilled Manual, Unskilled Manual, and Others was adopted from the Nigeria Demographic and Health Survey (NDHS) 2018 [ 21 ]. Occupation was recoded into three groups as Professional/Technical/Managerial/Clerical, Sales and Services/Skilled Manual and Unskilled Manual/Others because some categories have very few cases which may affect the stabilitity of the logistic model. Variable selection in the forward stepwise logistic regression was based on the Wald statistic, with variables entered if p  < 0.05. Results were reported as odds ratios (ORs) with 95% confidence intervals (CI), and statistical significance was set at p  < 0.05. To ensure the reliability of the logistic regression model, multicollinearity was assessed using Variance Inflation Factor (VIF) and tolerance statistics. All the predictor variables had VIF values below 2, indicating no significant multicollinearity. The tolerance values were also above 0.1, confirming that none of the independent variables were excessively correlated. The selection of a 25% DFI cut-off for distinguishing between good and poor sperm DNA integrity was guided by the Halosperm ® kit manufacturer’s recommendations and this aligned with thresholds used in previous studies using the SCD method [ 5 , 7 , 22 ]. Although tests like SCSA (Acridine Orange Flow Cytometry) typically apply 15%, 25%, and 40% thresholds, the SCD technique, including Halosperm ® , has often used similar cutoffs for clinical interpretation. It is important to note that other techniques such as the TUNEL assay and Comet assay have suggested alternative thresholds (usually 20%) based on analytical evidence [ 23 ]. This emphasizes the importance of method-specific cutoffs and standardization when comparing DNA fragmentation results across different assays. In this study, we carefully adhered to the recommended cutoff for the assay employed to ensure consistency and clinical relevance. This study was conducted with the by the State Ethics Committee on Health Research. Written consent was obtained from all the patients for the main study that generated the study data.

Table  1 presents demographic and lifestyle factors alongside the frequency distribution of individuals undergoing DFI (DNA Fragmentation Index) analysis. The participants are divided into two age groups: those below 45 years and those aged 45 years and above. The mean DNA fragmentation index (DFI) differed significantly across several demographic and lifestyle factors. Men under 45 years had a significantly lower mean DFI (35.30 ± 19.77%) compared to those aged 45 years and above (40.79 ± 20.57%), with the difference reaching statistical significance ( t = −3.44, p  = 0.001). In terms of occupational categories, marked variability in DFI was observed. Men in professional, technical, or managerial roles had the lowest mean DFI (29.64%), whereas those in sales and service (48.39%), skilled manual (49.88%), and other unspecified occupations (50.60%) had substantially higher mean values. Table 1 Demographic and lifestyle characteristics of participants undergoing DNA fragmentation index (DFI) at nordica fertility centre Variable N Mean DFI (%) SD t-value p -value Age (years)  < 45 358 35.30 19.77 −3.44 0.001  ≥ 45 285 40.79 20.57 Occupation  Professional/Technical/Managerial 368 29.64 19.54 - -  Clerical 2 29.00 4.24 - -  Sales and Services 209 48.39 16.28 - -  Skilled Manual 17 49.88 11.59 - -  Unskilled Manual 6 40.00 9.32 - -  Others 42 50.60 15.03 - - Alcohol Use/Drinking  Yes 279 36.92 20.11 0.44 0.657  No 338 37.64 20.22 Smoking  Yes 59 34.54 19.39 1.11 0.266  No 558 37.61 20.23 Frequency of ejaculation  Abnormal 22 46.82 22.33 2.29 0.022  Normal 590 36.84 19.96  Body Mass Index  Normal weight (< 25) 204 38.00 20.80 0.32 0.75  Overweight (≥ 25) 421 37.46 19.99 Demographic and lifestyle characteristics of participants undergoing DNA fragmentation index (DFI) at nordica fertility centre Alcohol consumption did not significantly affect DFI, with drinkers and non-drinkers showing comparable mean values (36.92% versus 37.64%, p  = 0.657). On the other hand, smoking status was not significantly associated with DFI, as smokers had a mean DFI of 34.54% and non-smokers had a mean of 37.61% ( p  = 0.266). Ejaculatory frequency showed a significant association with sperm DNA integrity. Participants with abnormal ejaculation frequency showed a higher mean DFI (46.82 ± 22.33%) than those with normal frequency (36.84 ± 19.96%), and this difference was statistically significant ( t  = 2.29, p  = 0.022) Lastly, body mass index (BMI) classification did not show a statistically significant association with DFI. Men with normal BMI had a mean DFI of 38.00% while those classified as overweight had a mean of 37.46% ( p  = 0.75). Several studies have demonstrated that sperm quality, including DNA integrity, declines significantly after the age of 45. This decline is primarily attributed to increased oxidative stress, which leads to higher levels of sperm DNA fragmentation and reduced reproductive potential. For instance, a study published in Biology found that approximately 80% of DNA fragmentation observed in sperm is due to oxidative stress, which can arise from aging, frequent infections, and other factors. Additionally, research has shown that sperm DNA fragmentation increases with age and is likely related to both defective spermatogenesis and increasing oxidative stress levels. These findings highlight the impact of advanced paternal age on sperm DNA integrity [ 24 , 25 ] (Fig. 1 ). Fig. 1 Distribution of Age, Alcohol Use, Smoking Status and Ejaculation Frequency among Participants Distribution of Age, Alcohol Use, Smoking Status and Ejaculation Frequency among Participants Semen parameters and DNA Fragmentation Index (DFI) were stratified by age group to explore age-related trends (Table  2 ). The mean sperm concentration showed minimal variation across groups, ranging from 18.2 ± 16.4 × 10⁶/ml in men aged 35–45 years to 21.9 ± 16.7 × 10⁶/ml in those under 35 years. Table 2 Semen parameters and DNA fragmentation index (DFI) across age groups among study participants Age Group (years) Sperm Concentration (10⁶/ml) Total Motility (%) Morphology (% normal) DFI (%)  45 20.5 ± 17.0 31.1 ± 20.3 10.4 ± 7.5 41.4 ± 20.5 Semen parameters and DNA fragmentation index (DFI) across age groups among study participants A progressive decline in total motility was observed with increasing age. Men younger than 35 years had the highest motility (43.5 ± 18.9%), while those over 45 years showed the lowest (31.1 ± 20.3%). Similarly, normal morphology percentages were highest among the 35–45 age group (13.0 ± 8.8%) and lowest in men older than 45 years (10.4 ± 7.5%). DFI increased with age, indicating greater sperm DNA damage among older participants. The mean DFI was 30.4 ± 16.7% in men under 35 years, rising to 41.4 ± 20.5% in men over 45 years, suggesting an age-associated decline in sperm DNA integrity. Table  3 presents a detailed analysis of the relationship between various demographic and lifestyle factors and the DNA Fragmentation Index (DFI) among infertility patients. The DFI levels are categorized into two groups as DFI ≤ 25% and DFI > 25%. The table includes the number and percentage of participants in each category, along with chi-square (χ 2 ) values and p-values to determine statistical significance. Table 3 Association of DNA fragmentation index (DFI) levels with demographic and lifestyle factors in fertility patients DFI ≤ 25% DFI > 25% N (%) N (%) χ 2 p -value Age (years)  < 45 148(64.9) 210 (50.6) 12.21 0.001  Above 45 80 (35.1) 205 (49.4) Occupation  Professional/Technical/Managerial 217 (59.0) 151 (41.0) 162.75 0.0001  Clerical 0 (0.0) 2 (100)  Sales and Services 10(4.8) 199 (95.2)  Skilled Manual 0 (0.0) 17 (100)  Unskilled Manual 1 (16.7) 5 (83.3)  Others 0 (0.0) 42 (100) Alcohol Use/Drinking  Yes 96 (46.2) 183 (42.0) 3.55 0.169  No 108 (51.9) 230 (52.8) Smoking  Yes 24 (10.8) 35 (8.9) 0.581 0.446  No 199 (89.2) 359 (91.1) Frequency of ejaculation  Abnormal 6 (2.7) 16 (4.1) 0.800 0.371  Normal 216 (97.3) 374 (95.9) Association of DNA fragmentation index (DFI) levels with demographic and lifestyle factors in fertility patients Among participants aged below 45 years, 64.9% had DFI levels of ≤ 25%, while 50.6% had DFI levels > 25%. In contrast, among those aged above 45 years, 35.1% had DFI levels ≤ 25%, and 49.4% had DFI levels > 25%. This difference was statistically significant, as indicated by the chi-square test (χ 2  = 12.21, p  = 0.001), suggesting that age is associated with DFI levels. Occupation significantly associated with DFI levels. Among professionals/technical/managerial workers, 59.0% (217 participants) had a DFI ≤ 25%, and 41.0% (151 participants) had a DFI > 25%. For those in clerical positions, all participants (2 participants) had a DFI > 25%. In the sales and services category, only 4.8% (10 participants) had a DFI ≤ 25%, while 95.2% (199 participants) had a DFI > 25%. Similarly, 100% of participants in skilled manual jobs (17 participants) and 83.3% (5 participants) in unskilled manual jobs had a DFI > 25%. The chi-square value (Chi-squared test for trend) of 162.75 and a p-value of 0.0001 indicate a highly significant relationship, with non-professional jobs associated with higher DFI levels. These findings indicate that individuals engaged in non-professional jobs exhibit significantly higher DFI levels compared to those in professional, technical, or managerial roles. Several mechanisms may explain this relationship, including occupational exposure to environmental toxins, chronic work-related stress, heat exposure, and irregular work schedules, all of which have been linked to impaired sperm quality and increased oxidative stress [ 26 , 27 ]. There was no statistically significant difference in DFI levels based on alcohol use. Among participants who reported drinking alcohol, 46.2% had DFI levels ≤ 25%, while 42.0% had DFI levels > 25%. Similarly, among non-drinkers, 51.9% had DFI levels ≤ 25%, and 52.8% had DFI levels > 25% (χ 2  = 3.55, p  = 0.169). The difference in DFI levels between smokers and non-smokers was not statistically significant. Among smokers, 10.8% had DFI levels ≤ 25%, and 8.9% had DFI levels > 25%. Among non-smokers, 89.2% had DFI levels ≤ 25%, and 91.1% had DFI levels > 25% (χ 2  = 0.581, p  = 0.45). However, data on duration and intensity of alcohol consumption and smoking habit was not collected but alcohol and smoking were measured categorically (yes/no) in this study. There was no statistically significant association between frequency of ejaculation and DFI levels. Among participants with abnormal ejaculation frequency, 2.7% had DFI levels ≤ 25%, while 4.1% had DFI levels > 25%. In contrast, among those with normal ejaculation frequency, 97.3% had DFI levels ≤ 25%, and 95.9% had DFI levels > 25% (χ 2  = 0.800, p  = 0.37). These findings suggest that age and occupation are associated with DFI levels, while alcohol use and smoking habits showed weaker associations. Understanding these associations can help in identifying potential risk factors and guiding interventions aimed at improving sperm DNA integrity and fertility outcomes. The results presented in Table  4 dwell on DFI levels based on medical conditions, PICSI Binding, Motility, Number of Embryos Transferred and Pregnancy Outcome, respectively. Table 4 DNA fragmentation index (DFI) with medical conditions, PICSI binding, sperm motility, and pregnancy outcomes DFI ≤ 25% DFI > 25% N (%) N (%) χ 2 p -value Medical Conditions  Diabetes 11 (5.0) 20 (5.2) 0.211 0.646  Diabetes and hypertension 0 (0.0) 1 (0.3)  Hypertension 30 (13.8) 59 (15.4)  Others 10 (4.6) 16 (4.2)  None 167 (76.6) 288 (75.0) PICSI Binding  No binding 46 (22.5) 74 (21.0) 3.497 0.061  Very poor binding 11 (5.4) 47 (13.4)  Poor binding 37 (18.1) 85 (24.1)  Fair binding, 95 (46.6) 132 (37.5)  Adequate binding 15 (7.4) 14 (4.0) Motility  Poor 2 (0.9) 18 (4.3) 5.724 0.017  Fair 219 (96.1) 390 (93.8)  Good 7 (3.1) 8 (1.9) Number of embryos transferred  0–1 65 (31.7) 114 (31.8) 0.001 0.991  2–3 140 (68.3) 245 (68.2) Mean (SD) 1.89 (1.14)  Pregnancy Outcome  Positive 40 (17.5) 52 (12.5) 5.346 0.069  Negative 134 (58.8) 236 (56.7) DNA fragmentation index (DFI) with medical conditions, PICSI binding, sperm motility, and pregnancy outcomes Among those with DFI ≤ 25%, 11 participants (5.0%) had diabetes, compared to 20 participants (5.2%) with DFI > 25%. For diabetes and hypertension, there were no participants (0.0%) with both conditions in the DFI ≤ 25% category, and only 1 participant (0.3%) in the DFI > 25% category. In the DFI ≤ 25% group, 30 participants (13.8%) had hypertension, while 59 participants (15.4%) in the DFI > 25% group had hypertension. For other medical conditions, there were 10 participants (4.6%) with other medical conditions in the DFI ≤ 25% group and 16 participants (4.2%) in the DFI > 25% group. A large majority of participants reported no medical conditions, with 167 participants (76.6%) in the DFI ≤ 25% group and 288 participants (75.0%) in the DFI > 25% group. The association was not statistically significant (χ² = 0.211, p  = 0.646). A larger proportion of participants with DFI > 25% demonstrated suboptimal binding capacity. Specifically, very poor and poor binding were more common among those with elevated DFI (13.4% and 24.1%, respectively) compared to the lower DFI group (5.4% and 18.1%). Conversely, fair binding and adequate binding were more frequently observed among those with DFI ≤ 25% (46.6% and 7.4%) than their counterparts with higher DFI (37.5% and 4.0%), respectively. The overall association between PICSI binding and DFI was not statistically significant (χ² = 3.497, p  = 0.061). Sperm motility was analyzed and categorized as poor, fair, or good. Only 2 participants (0.9%) with DFI ≤ 25% had poor motility, compared to 18 participants (4.3%) with DFI > 25%. The majority of participants fell into the fair motility category, with 219 participants (96.1%) in the DFI ≤ 25% group and 390 participants (93.8%) in the DFI > 25% group. Good motility was observed in 7 participants (3.1%) with DFI ≤ 25% and 8 participants (1.9%) with DFI > 25%. In terms of sperm motility, a statistically significant difference was found between the groups (χ² = 5.724, p  = 0.017). For participants who had 0–1 embryos transferred, 65 participants (31.7%) were in the DFI ≤ 25% group while 114 participants (31.8%) were in the DFI > 25% group. For participants who had 2–3 embryos transferred 140 participants (68.3%) were in the DFI ≤ 25% group while 245 participants (68.2%) were in the DFI > 25% group. The number of embryos transferred (categorized as 0–1 vs. 2–3) did not differ significantly between the DFI groups (χ² = 0.001, p  = 0.991). The mean number of embryos transferred was 1.89 ± 1.14. The pregnancy outcomes for participants were also examined. There were 40 participants (17.5%) with a positive pregnancy outcome in the DFI ≤ 25% group, compared to 52 participants (12.5%) in the DFI > 25% group. A negative pregnancy outcome was observed in 134 participants (58.8%) with DFI ≤ 25% and 236 participants (56.7%) with DFI > 25%. The results showed that clinical pregnancy was more frequently observed in the lower DFI group (17.5%) compared to the higher DFI group (12.5%), although this trend was not statistically significant (χ² = 5.346, p  = 0.069). The final forward stepwise logistic regression model analysis in Table  5 shows that age above 45 years (OR = 2.45, 95% CI: 1.39–4.32, p  = 0.002), occupation category (Professional/Technical/Managerial/Clerical) (OR = 0.001, 95% CI: 0.0001–0.011, p  < 0.001), and alcohol use (OR = 18.01, 95% CI: 7.03–46.12, p   25%) Variable Β* SE Wald p -value OR (Exp(B)) 95% CI for OR Age (years)  ≥45 0.896 0.29 9.58 0.002 2.45 1.389–4.322 Occupation  Professional/Technical/Managerial/Clerical −6.746> 1.13 35.92 < 0.001 0.001 0.0001–0.011  Sales and Services/Skilled Manual −1.218 1.07 1.29 0.257 0.296 0.036–2.429  Drinking 2.891 0.48 36.28 < 0.001 18.006 7.030–46.123  Constant 3.203 1.02 9.83 0.002 24.608 * Β Regression Coefficient, SE Standard Error, OR  Odds Ratio Multivariable logistic regression analysis of factors associated with high DNA fragmentation index (DFI) (> 25%) * Β Regression Coefficient, SE Standard Error, OR  Odds Ratio

Male infertility is defined as the inability to conceive after 12 months of regular, unprotected sexual intercourse. While alterations in sperm parameters such as concentration, motility, and morphology are associated with infertility, they are not solely used to define the condition [ 1 ]. Causes of male infertility include congenital or acquired urogenital abnormalities, malignancies, increased scrotal temperature from varicoceles, endocrine disturbances, genetic abnormalities and immunological factors [ 2 ]. These issues often prompt men to seek medical help to raise a family [ 3 ]. Seminal fluid analysis has been used to assess male fertility for a long time but some individuals with normal semen parameters fail to achieve conception even with assisted reproductive technology. The advent of sperm DFI has resolved some of these challenges as some of these problems have been found to be due to defective DNA [ 4 ]. The relationship between sperm DNA fragmentation and infertility was first introduced over 40 years ago by Dr Donald P Evenson. The first DNA fragmentation test was the Sperm Chromatin Structure Assay (SCSA) which was also introduced at the same time by Dr Donald P. Evenson [ 5 , 6 ]. Sperm DNA fragmentation refers to breaks in the single and double strands of DNA in the male gamete and this can lead to failure of fertilization, poor embryonic development, failure to achieve a clinical pregnancy, miscarriage and recurrent pregnancy loss [ 7 – 9 ]. The DNA fragmentation can occur at various stages during spermatogenesis. Apoptosis occurs during spermatogenesis to remove abnormal and excess sperm cells. The action of endonuclease enzymes that cleave DNA during apoptosis leads to the release of defective spermatozoa with high levels of fragmented DNA [ 6 ]. Torsional stress during sperm maturation can cause breaks in the DNA. Immature chromatin which is not fully compact or is decondensed make sperm DNA more susceptible to damage by various insults [ 7 ]. Sperm DNA fragmentation has become an important indicator in assessing male infertility, as it provides valuable information about the genetic quality of sperm that standard semen analysis may not fully capture. Several intrinsic and extrinsic factors, including age, oxidative stress, and ejaculatory patterns, have been linked to elevated levels of DNA fragmentation. Ejaculation frequency and latency (i.e., the time between ejaculations) are increasingly recognized as modifiable lifestyle factors influencing sperm quality and DNA integrity. Longer ejaculatory abstinence periods have been associated with increased sperm DNA damage due to prolonged exposure to reactive oxygen species within the epididymis. Understanding the relationship between ejaculation timing and DFI is therefore important for developing practical recommendations for men undergoing fertility evaluation or treatment. The risk factors for sperm DNA fragmentation include varicocele, male genital tract infection, medical conditions, e.g., diabetes, obesity, malignancies, advanced male age, exposure to radiation, chemicals toxins, lifestyle of smoking, alcohol, use of marijuana [ 8 – 10 ]. The main Sperm DNA fragmentation detection methods include Sperm Chromatin structure assay (SCSA), Sperm chromatin diffusion method (SCD), COMET assay (CA) and Terminal deoxyuridine nick end labelling (TUNEL) [ 11 , 12 ]. In 2021, sperm DFI test became the first evidence- based test to be included in the international guideline [ 13 – 15 ]. Very few studies have been done on sperm DFI in Nigeria [ 16 , 17 ]. In addition, this study includes PICSI (Physiological Intracytoplasmic Sperm Injection) binding analysis, a functional sperm selection method based on sperm binding to hyaluronic acid (HA). This assay mimics the natural sperm selection process in the female reproductive tract and allows for the selection of mature, biochemically competent spermatozoa with lower DNA fragmentation levels. A strong HA-binding capacity has been associated with improved fertilization and embryo development outcomes in assisted reproduction. Thus, assessing the correlation between PICSI binding scores and DFI could offer deeper understanding of the functional quality of sperm in this population. This study aimed to analyze the demographic and lifestyle factors associated with DFI in a cohort of male patients seeking fertility evaluation at the Nordica Fertility Centre [ 18 ]. Exploring associations between DFI levels and various demographic, lifestyle, and medical factors is with a view to better understanding their impact on sperm DNA integrity and fertility outcomes. This study is among the first in Nigeria to evaluate the association between DFI, ejaculation characteristics, and PICSI binding ability in a cohort of male patients presenting for fertility evaluation.

The results of the study indicated that demographic and lifestyle factors have significant associations with sperm DNA integrity, as measured by the DNA Fragmentation Index (DFI). The majority of the study participants were below 45 years old (55.7%), with an average age of 44.1 years. This age distribution is important as it correlates with various fertility parameters, including sperm quality and DNA integrity. The data showed a statistically significant association between age and DFI levels, with older age linked to higher rates of poor sperm DNA integrity (χ² = 12.21, p  = 0.001). This finding is consistent with previous research that suggests sperm DNA fragmentation increases with age, likely due to accumulated environmental exposures and a decline in cellular repair mechanisms [ 28 , 29 ]. In terms of occupation, individuals in professional, technical, or managerial roles constituted the largest group (57.1%), and these participants generally exhibited better sperm DNA integrity compared to those in clerical, sales and services, or manual laour positions. The significant association between occupational stress, lifestyle habits, and exposure to environmental toxins. Similar patterns have been observed in studies that link occupational stress and exposure to poor sperm quality [ 29 , 30 ]. Alcohol consumption was reported by 45.2% of the participants. Although there was no statistically significant difference in DFI levels based on alcohol use (χ² = 3.55, p  = 0.169), the distribution of sperm DNA integrity categories suggests a trend where alcohol users have higher rates of poor sperm DNA integrity. This aligns with the understanding that alcohol can induce oxidative stress and damage sperm DNA [ 31 ]. The difference in DFI levels between smokers and non-smokers was not statistically significant though may be clinically important. Among smokers, 10.8% had DFI levels ≤ 25%, and 8.9% had DFI levels > 25%. Among non-smokers, 89.2% had DFI levels ≤ 25%, and 91.1% had DFI levels > 25% (χ 2  = 0.581, p  = 0.45). This finding is supported by extensive literature documenting the detrimental effects of smoking on sperm quality and DNA integrity due to oxidative stress and the presence of toxic substances in cigarette smoke [ 32 ]. The frequency of ejaculation was found not to be significantly associated with sperm DNA integrity. Participants with normal ejaculation frequency (96.6%) had better sperm DNA integrity compared to those with abnormal ejaculation frequency (χ² = 0.800, p  = 0.37). Notably, none of the participants with abnormal ejaculation frequency had excellent to good sperm DNA integrity, and half of them exhibited very poor sperm DNA integrity. Understanding these associations can help in identifying potential risk factors and guiding interventions aimed at improving sperm DNA integrity and fertility outcomes [ 33 , 34 ]. Normal ejaculation frequency in this study is referred to as frequency of ejaculation between three to four times a week while frequency of ejaculation below this is defined as abnormal [ 35 ]. Medical conditions such as diabetes and hypertension were also analyzed for their association with DFI levels. Although no statistically significant association was found (χ² = 0.211, p  = 0.646), participants with these conditions tended to have higher DFI levels, which may indicate a potential impact of systemic health on sperm DNA integrity. Chronic conditions like diabetes and hypertension are known to affect overall cellular health, including that of germ cells [ 36 , 37 ]. The findings indicate that elevated DFI levels are associated with poorer sperm motility and weaker PICSI binding capacity. These associations suggest a potential link between sperm DNA integrity and functional sperm parameters such as hyaluronic acid binding which is an essential feature for selecting mature and competent spermatozoa. Although the association between PICSI binding and DFI was not statistically significant, the trend supports previous literature highlighting reduced binding ability in sperm with high DNA fragmentation [ 38 ]. Sperm motility showed a significant association with DFI levels. Poor motility was more prevalent among those with higher DFI, reinforcing the link between motility and sperm DNA integrity. Good motility is essential for natural conception and successful ART outcomes, and its association with lower DFI highlights the importance of comprehensive sperm assessments in fertility treatments [ 39 ]. Importantly, while the number of embryos transferred did not vary by DFI group, pregnancy rates appeared lower among individuals with higher DFI, though the difference was not statistically significant. These observations emphasize the potential negative impact of sperm DNA fragmentation on reproductive outcomes, aligning with prior studies that demonstrate diminished pregnancy rates with elevated DFI. This finding can be linked to the study by Wang et al. from January 1 st 2017 to March 31 st 2022 [ 40 ]. Pregnancy outcomes were analyzed in relation to DFI levels, revealing that higher DFI levels were associated with lower positive pregnancy rates, though the association was not statistically significant. This trend suggests that sperm DNA fragmentation might negatively impact fertilization, embryo development, and implantation rates, which is supported by literature indicating that high sperm DNA fragmentation can compromise pregnancy outcomes in ART [ 41 , 42 ]. Our findings indicate that older age and non-professional occupations are significantly associated with higher DFI levels in men seeking fertility evaluation. These results are consistent with previous studies reporting an age-related decline in sperm DNA integrity [ 43 , 44 ]. The association between occupation and DFI might be attributed to occupational exposures to environmental toxins or lifestyle factors associated with certain professions [ 45 ]. While the study did not find a significant association between DFI and alcohol use or smoking habits, this could be due to limitations in self-reported data or the retrospective nature of our study. Further research with larger sample sizes and prospective data collection is needed to confirm these findings. Importantly, we found a significant association between sperm motility and higher DFI levels, highlighting the interconnectedness of these sperm parameters [ 46 ]. The trend towards significance in pregnancy outcomes suggests that elevated DFI might negatively impact reproductive success. The forward stepwise logistic regression model analysis showed that age above 45 years (OR = 2.45, 95% CI: 1.39–4.32, p  = 0.002), occupation category (Professional/Technical/Managerial/Clerical) (OR = 0.001, 95% CI: 0.0001–0.011, p  < 0.001), and alcohol use (OR = 18.01, 95% CI: 7.03–46.12, p  < 0.001) were independently associated with high high sperm DNA fragmentation index (DFI). The logistic regression model thus predicts the likelihood of being in the high DFI group based on variables like age, occupation and alcohol use. This research has several limitations that should be considered when interpreting the findings of the study. First, its retrospective design may introduce recall bias, particularly for self-reported lifestyle factors such as alcohol consumption, smoking habits, and occupational exposures. Participants may underreport or overreport behaviours due to social desirability bias or memory inaccuracies, which could affect the validity of the associations observed. Past studies have highlighted the challenges inherent in relying on self-reported lifestyle data in reproductive health research [ 47 ]. Hormonal profiles such as FSH, LH and testosterone were not available for all participants and thus were not uniformly analyzed and female partner’s characteristics, such as age, hormonal profile, and reproductive history, were not included in the analysis, which limits the ability to fully interpret embryo transfer and pregnancy outcomes. Additionally, while multiple potential confounders were adjusted for in the analysis, residual confounding cannot be completely excluded. Factors such as dietary habits, psychological stress, environmental exposures, and genetic predisposition, which were not captured in this study, may also influence sperm DNA fragmentation [ 48 ]. Moreover, the lack of longitudinal follow-up limits our ability to assess causal relationships between lifestyle, demographic factors and DFI changes over time. Prospective studies with repeated measures are needed to strengthen the evidence base. Finally, although the SCSA method used for DFI measurement is widely validated, it is important to acknowledge inherent methodological constraints, including inter-laboratory variability and the potential influence of sample handling on DFI results. Future research should consider multicenter standardization efforts and comparative evaluations across different DFI assays to enhance generalizability.

This study provides valuable insights into the factors associated with DFI in a cohort of male fertility patients. Our findings emphasize the importance of considering factors like age, occupation, and sperm motility during fertility assessments. Early identification of individuals with elevated DFI and potential risk factors is crucial for guiding appropriate interventions, such as lifestyle modifications or assisted reproductive technologies, to improve chances of successful conception. While alcohol use and medical conditions did not show statistically significant associations, their potential impact on sperm DNA integrity warrants further investigation. The relationships between PICSI binding, sperm motility, and DFI levels highlight important considerations for selecting sperm in ART. Lastly, the trend toward lower pregnancy success rates with higher DFI levels emphasizes the clinical relevance of sperm DNA fragmentation in fertility treatments. Future studies should continue to explore these associations and develop strategies to mitigate the adverse effects of these factors on sperm DNA integrity.