Does sperm DNA fragmentation correlate with semen parameters?

Abstract Purpose This study aimed to investigate the association between sperm quality assessed by routine semen analysis and sperm DNA integrity assay. Methods In our cross‐sectional study, a total of 318 men from the infertile couples were enrolled from December 2017 to March 2019 at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam. General characteristics and semen parameters were detected. The sperm DNA fragmentation index (DFI) was estimated by the sperm chromatin dispersion (SCD) assay. A threshold of DFI 30% was applied to classify normal (DFI < 30%) or abnormal (DFI ≥ 30%) groups. The correlations between DFI and semen parameters were analyzed by Spearman's rank correlation coefficient. Results In the correlation analysis, DFI was significantly correlated with abnormal head and progressive motility, with a positive correlation with abnormal head (ρ = .202, P = .0003) and a weak negative correlation with progressive motility (ρ = −.168, P = .0027), respectively. In the bivariate analysis, DFI was associated with male age, smoking, and alcohol consumption with P < .05. Conclusions The sperm DFI was not strongly correlated with conventional semen parameters. Therefore, a sperm DNA fragmentation assay should be performed as an additional step in the investigation of male fertility.

DNA fragmentation is expressed as the DNA fragmentation index (DFI). DNA fragmentation rates often correlate with semen analysis parameters through a high abnormal DFI (>30%) and may be found in up to 8% of infertile men with a normal semen analysis, suggesting an adjunct role for the standard semen analysis. 5 In studies of natural pregnancy rates stratified by DFI, the rates of conception were statistically lower among couples with an elevated DFI.
In recent years, a number of tests were introduced for the evaluation of sperm chromatin structure, including TUNEL (terminal dUT-Pnick-end labeling), the COMETtest (single cell gel electrophoresis), the AO (acridine orange) test, the CMA3 (chromomycin A3) test, the SCSA (sperm chromatin structure assay) test, and the SCD (sperm chromatin dispersion) test. 6,7 Except SCD test, most fragment DNA sperm tests require advanced equipments and high cost. The difference between fragmented and nonfragmented sperm was distinguished from conventional microscopes. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with nonfragmented DNA. 8 The SCD test is a very simple, rapid, and accurate procedure to determine SDF. 9 In addition, the SCD test method was equivalent or more sensitive to analysis sperm DNA fragmented than the TUNEL method. 2 Deproteinized nuclei create the halos of dispersed DNA that correspond to relaxed DNA loops attached to the residual nuclear structure nuclei with fragmented DNA that produce either small halos or no halos of dispersed DNA.
In contrast, the sperm nuclei without DNA fragmentation release DNA, creating large halos, or slightly fragmented DNA, creating medium halos. 10 By routine semen analysis, the normal parameters, including sperm concentration, motility, and morphology, does not ensure normal sperm DNA. However, fertilization can occur even with damaged DNA, resulting in subsequent unsuccessful pregnancy outcomes. The relationship between sperm morphology and the degree of sperm DNA damage has not yet been clearly understood. This study aimed to investigate the association between sperm quality assessed by routine semen analysis and sperm DNA integrity assay.

| Clinical approaches
General information was recorded regarding age, occupation, geography, infertility duration, type of infertility, history of internal diseases or surgery, smoking, and alcohol resumption. Physical examination was performed to measure the BMI, waist-hip circumference, and male genital examination concerning any abnormalities in the penis, scrotum, and testis.

| Semen analysis
A semen sample was collected and analyzed following the WHO standard 2010. 3 Microscopic examination assessed the sperm motility, vitality, concentration, and sperm morphology.

Sperm motility
The sperm motility parameter was analyzed by manual counting under phase-contrast microscopy (Primo Star, Zeiss) at 400x total magnification. The sperm motility is of two categories: progressive motility and nonprogressive motility. In this study, the progressive motility of 200 sperms was assessed.

Sperm vitality
The vitality parameter was assessed by the eosin technique under phase-contrast microscopy (Primo Star, Zeiss) at 400× total magnification as recommended by the WHO. Two hundred cells were counted immediately following liquefaction of the semen samples, and the percentage of viable cells was calculated.

Sperm morphology
This parameter was estimated by Giemsa staining. The morphology of sperm head shape and size, acrosomal region, sperm neck, midpiece, tail, and cytoplasmic droplets was determined under microscopy (Zeiss) at 1000× total magnification, according to the 5th edition of the WHO guidelines. At least, 200 sperms were counted to calculate the percentage of both normal and abnormal morphology.

| Statistical analysis
The studied population of men was divided into two groups regarding the DFI value. Based on the recommendation of Halosperm® provided by Halotech (Halotech DNA SL), the DFI threshold was fixed at 30% to distinguish between two groups: DFI ≥ 30% group and DFI < 30% group. This threshold has been used by other authors, who have indicated that DFI levels above 30%, as measured by SCD and sperm chromatin structure assay (SCSA), showed a negative correlation between sperm DNA fragmentation and semen parameters. 11,12 All statistical analyses were performed using SPSS software The Wilcoxon rank sum test and Kruskal-Wallis test were applied to compare the median value for two groups or several groups.
Differences between the values were considered statistically significant when P < .05.

| RE SULTS
A total of 318 men in infertile couples were recruited for the study group.  TA B L E 1 General characteristics of study population and DNA fragmentation index negative correlation between DFI and progressive motility (ρ = −.168, P = .0027).

| D ISCUSS I ON
Sperm DNA damage is the major molecular cause of male infertility, which has a negative effect on reproductive outcomes in couples.
Recent clinical practice recommendations suggest the potential role of sperm DNA fragmentation assay in specific clinical scenarios.
This would expand the potential of sperm DNA fragmentation assay globally as a prognostic and diagnostic tool in various male infertility scenarios and their treatment management. 13 Semen analysis by testing conventional parameters is the primary method for assessing men fertility, according to WHO guidelines. It is clear that routine semen analysis can provide a limited prediction of man fertility potential and is not always able to explain the cause of male infertility. In fact, many cases of male infertility are caused by sperm DNA defects, which routine semen quality analyses fail to detect. 14 20 The rolling of sperm DNA materials is mediated by specific proteins that control DNA condensation and decompression over time.
DNA must be compressed to be protected from degeneration and fragmentation before decompression to reveal vertical frames for protein synthesis at embryonic development stages. 21 Spermatozoa from the infertile men group exhibited a high proportion of DNA damage. 22 Some reports showed a statistically significant correlation between the sperm DNA fragmentation rate (SDC assay) and the following sperm characteristics: sperm motility, morphology, and concentration. 15,23 Sivanarayana et al reported that sperm with DNA fragmentation showed a negative correlation with semen parameters: concentration, motility, and normal morphology were significantly lower in the abnormal DNA group (DFI ≥ 30%) than in the normal DNA group (DFI < 30%). 11  In contrast, other studies found no statistically significant correlations between the conventional semen parameters and the degree of sperm DNA fragmentation. 17,18 Even in men with oligozoospermia, there were no significant correlations between sperm DNA fragmentation and progressive motility, concentration, or morphology. 25 DFI was correlated with only one of the parameters, such as morphology 26,27 or motility. 25 Our results in men from infertility couples showed no correlation between DFI and volume, concentration, or vitality, but DFI and progressive motility showed a negative correlation.
Regarding sperm morphology, head abnormalities, especially amorphous heads, are reported to be related to the elevated degree of DNA fragmentation. 28 The percentages of normal nuclear sperm showed a significant negative correlation with the percentage of DNA fragmentation. 29 By evaluating sperm head shape using el-  31 Negative effects at this stage will lead to the abnormal morphology of spermatozoa and DNA damage but may not affect the neck-tail. Therefore, DFI may be related to sperm head abnormalities but is not related to neck-tail abnormalities.
Epigenetic changes and DNA mutations along with chromosomal aneuploidies have been associated with increasing paternal age. 32 The conventional semen analysis can often fail to detect a defect in spermatogenesis (high DFI) in older men and suggest that infertile couples with advanced paternal age, including those with normal semen parameters, should consider sperm DNA testing as part of the couple evaluation. 33,34 In the present study, we found a significant difference between sperm DNA fragmentation based on age in the two groups (≥35 years vs <35 years). However, in the multivariate analysis, the correlation between DFI and age was not statistically significant (r = .095, P = .093).
Stress and lifestyle may affect sperm DNA damage.. In clinical studies, the relationship between smoking and sperm DNA fragmentation has been discussed in controversial conclusions. 17,35 In this study, we found a significant effect of smoking on sperm DNA fragmentation with the DFI of smokers compared with nonsmokers: 23.72 ± 16.16 compared with 20.11 ± 18.20.
Human studies have revealed that alcohol consumption causes significant morphological changes in sperm, leading to abnormal head and tail sperm. 36 A study by Komiya et al showed that the DFI was significantly different based on alcohol status, and chronic alcohol use increased the DFI by 49.6 ± 23.3% compared with 33.9 ± 18.0% in those who did not regularly consume alcohol (P = .0084). 35 Our results showed that the DFI (23.49 ± 18.03) was increased in the semen samples from those with alcohol use (n = 142) compared with those who did not regularly consume alcohol (19.91 ± 16.94) (P = .0132).
In conclusion, our data showed that the sperm DNA fragmentation index was not strongly correlated with conventional semen parameters. Therefore, the sperm DNA fragmentation assay should be performed as an additional step in the investigation of male fertility.
Assessment of sperm fragmentation DNA is especially necessary for advanced age patients and men with risk factors such as smoking, alcohol exposure, or sperm with high rate of abnormal head.