Effects of adverse semen parameters on total oxidation status and DNA/chromatin integrity

Infertility is a major health problem that affects 7% of the men’s population. Oxidative stress (OS) plays a significant role in the pathophysiology of male infertility. The purpose of this study comparatively evaluated the total anti-oxidation status and DNA/chromatin integrity in semen samples among different infertile men’s groups compared with the normozoospermic men. This cross-sectional study contains four experimental groups, including teratozoospermia (Exp I), asthenoteratozoospermia (Exp II), oligoasthenoteratozoospermia (Exp III), and azoospermia (Exp IV), as well as the control group of normozoospermic men. The total antioxidant capacity (TAC) and total oxidant status (TOS) were assessed by applying the enzyme-linked immunosorbent assay. The chromatin/DNA damage was assessed in semen samples of all study groups by applying chromomycin A3 (CMA3) and toluidine blue (TB) staining methods. The results showed significantly higher proportions of TB+ and CMA3 positive sperm in all experimental groups compared to controls (P < 0.001). TAC, TOS, and the ratio of TAC to TOS were significantly different in all experimental groups compared to the normozoospermic men (P < 0.001). Our study demonstrated that at least one sperm parameter abnormality, such as teratozoospermia could cause serious defects at the levels of DNA/chromatin as well as the antioxidants to oxidant balance of human spermatozoa in subfertile men with abnormal spermogram. Infertile men with sperm morphological abnormalities may benefit from simultaneous assessment of sperm DNA defects and OS.

for fertilization and decreases sperm motility [9].OS can affect the mitochondrial copy number, mitochondrial DNA deletion, DNA methylation, and other DNA damage in spermatozoa, which affects semen quality and can be used as a factor to determine male fertility potential [10,11].The oxidative stress index (OSI) is a rapid, facile, and inexpensive indicator to determine the oxidant/ antioxidant ratio in seminal plasma and serum accurately.The OSI offers an invaluable and objective assessment of redox status [12].
Sperm DNA integrity is vital to the successful fertilization process and the transfer of the parental genetic content.Impaired chromatin packaging can cause the production of free radicals, which indirectly leads to DNA backbone break by decreasing protamine and forming disulfide bonds, as the oxidative attack seems to cause poor sperm morphology [13].Men with abnormal sperm parameters show more degrees of DNA damage [14].The decreased seminal antioxidants may be a main factor in the process of death associated with DNA damage in teratozoospermic men [15].Men with teratozoospermia may have a higher risk of sperm DNA break [16].Infertile men with isolated teratozoospermia (iTZS) had higher neutrophil-to-lymphocyte ratio (NLR) than normozoospermic men and increased sperm DNA fragmentation (SDF) values than an infertile participant with isolated azoospermia, isolated oligozoospermia, or normal semen parameters [17].Abnormal morphology can be caused by damage to sperm DNA, defects in chromatin density, and related unsuccessful pregnancy outcomes.The evidence highlighted the role of OS and SDF in the emergence of sperm with poor morphology [18].The main aim of our study was to assess the differences in sperm parameters, total antioxidant capacity (TAC), total oxidant status (TOS), OSI, and chromatin quality in men with different sperm parameters and whether the presence of one sperm parameter abnormality such as teratozoospermia could affect both OS and SDF in subfertile men with variable sperm parameters.

Study population
In this cross-sectional study, patients were divided into five groups, which include four experimental groups and a control group.The experimental groups included 15 teratozoospermic men (Exp I, T), 13 asthenoteratozoospermic men (Exp II, AT), 13 oligoasthenoteratozoospermic men (Exp III, OAT), and 18 azoospermic men (Exp IV).Also, the control group included 22 fertile men with normal sperm parameters (Fig. 1).In this Fig. 1 The schematic view of study design study, the semen samples were collected from 81 participants referred to Yazd reproductive sciences institute.The inclusion criteria of the control group were having normal sperm parameters and at least one child in two previous years, body mass index (BMI) ≤ 25, no history of smoking, no varicocele disease and no drug consumption, and age < 40.In the experimental groups, the inclusion criteria were the presence of BMI ≤ 25, no varicocele and infection disease and diabetes, no history of smoking, no drug consumption, no alcohol consumption, and age < 40.Exclusion criteria in all study groups included people with Pyospermia, fever, infectious diseases in the last three months, genetic problems, any infections or inflammatory diseases in the reproductive tract, sexually transmitted diseases, or erectile disorder.Our study was approved by the Ethics Committee of Shahid Sadoughi University, Yazd, Iran with approval code: IR.SSU.SPH.REC.1399.004.The consent form was signed by all participants before entering the study.

Semen analysis
The sample collection method was by masturbation with 2-5 days of sexual abstinence.In the liquefaction process, samples were incubated at 37 °C for 20 min.All assessments of sperm parameters were done based on the World Health Organization 2021 criteria.For evaluating sperm motility, a hundred spermatozoa were assessed by a phase-contrast Microscope with × 400 magnifications.The percentages of progressive, non-progressive, and immotile spermatozoa were analyzed according to WHO [10].The Diff-Quik staining kit (FaradidPardaz Pars Inc., Iran) was used to assess sperm morphology.The smears were stained according to the kit protocol.After that, 200 spermatozoa were evaluated by light microscopy at ×1000 magnification.The normal sperm percentage was recorded based on WHO [10].

Assessments of oxidative stress index
To evaluate the concentrations of total oxidant level (TOS) and total antioxidant level (TAC), we separated the seminal plasma of the samples from sperm cells by centrifugation at 1800 g for 10 min.The supernatant was applied to evaluate the OSI in seminal plasma.The OSI is computed by dividing TOS by TAC.The commercial kits (Naxifer TM, Navand, Salamat Co., Urmia, Iran) were applied to quantify the TOS and TAC.For preparing the samples, 10 7 cells of seminal plasma were washed with PBS and then homogenized with 1 ml Lysing Buffer.After centrifugation at 10,000 rpm for 10 min, the supernatant was separated and used as a sample.The reagents were placed at room temperature 30 min before use.If crystals were observed, the solution was homogenized by vortexing.For the TAC assay, after the preparation, reagent 2 was mixed with reagent 3 (1:1) and vortexed.Then, fivefold the volume of the prepared solution, R1 solution was added.The final solution was used as a working solution.5 µl of the sample/standard were poured into the wells of the plate (all standards and samples were assayed in duplicate), followed by adding 250 µl of the working solution.After 5 min, maximum absorption was recorded by a plate reader at 593 nm.For the TOS assay, after the preparation of the working and standard solution according to kit protocol, 30 µl of the sample/standard were added to the wells.200 µl of reagent 1 and 10 µl of reagent 2 was added to the wells and incubated for 20 min.After 20 min, maximum absorption was recorded by a plate reader at 530 nm.By using statistical software such as Excel, the standard curve graph of different concentrations was obtained and according to the line formula, the TAC and TOS were calculated.

Sperm DNA/chromatin integrity
After sperm analysis, each semen sample was smeared for toluidine blue (TB) and Chromomycin A3 (CMA3) staining.To assess sperm DNA/chromatin integrity, TB and CMA3 staining were performed to determine the chromatin damage and protamine deficiency of each sample.

Toluidine blue staining
In the first step, smears were prepared and dried.Then, they were fixed in 96% ethanol-acetone (1:1) at 4 °C for 30 min.0.1 Na HCl was used to hydrolyze samples at 4 °C for 5 min.The samples were washed with distilled water and then stained with 0.05% TB for 10 min at room temperature.In the last step, 200 sperm cells were evaluated at ×1000 magnification with light microscopy.Normal spermatozoa heads were stained pale blue, and abnormal spermatozoa heads were stained dark blue or purple.Abnormal spermatozoa percentage (TB + ) was recorded [19].

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) version 20 (IBM, California, United States) was used.To assess the normality of the data, Shapiro-Wilk test was performed.Data were expressed as mean ± SEM.Variables were analyzed using the Kruskal-Wallis variance analysis test and the Mann-Whitney U test.In addition, Spearman's test was applied to calculate the correlation coefficient.P < 0.05 was considered statistically significant.

Results
Every single one of the subjects was national Iranian, with a total mean (± SD) age of 33.78(± 5.29) years and a total mean (± SD) BMI of 24.29 (± 3.24).The age and BMI of all contributors were well-adjusted among all groups.Age and BMI did not show any significant differences (P ≥ 0.05).
Semen concentration and sperm morphology were significantly higher in the normozoospermic group compared to the other four groups (P < 0.001).The sperm normal morphology significantly declined in the infertile men with Teratozoospermia compared to the Oligoasthenoteratozoospermia group (P < 0.01).As expected, the percentages of immotile and motile sperms were respectively higher and lower in the II and III experimental groups compared to the controls (P < 0.001).Semen volume analysis did not show any difference that is worthy of attention.The semen analysis data regarding azoospermic samples were excluded due to the lack of sperm (Table 1).
The chromatin integrity data demonstrated significantly higher proportions of TB + and positive CMA3 spermatozoa in different sperm parameter variations of the experimental groups compared to the controls (P < 0.001).
There was a significant decrease in seminal plasma TAC levels in all experimental groups compared to the controls (P < 0.001).Moreover, TOS concentration significantly increased in the experimental groups compared to the normozoospermia group (P < 0.05).The OSI was almost in the balance range in the control group (OSI = 0.78 ± 0.09), while it was significantly higher (OSI = 2.77 ± 0.41) in all seminal plasma samples of the experimental groups (P < 0.001) (Table 2).
Sperm concentration, progressive motility, and normal morphology were negatively correlated with CMA3 + , TB + , and TOS levels.There was a significant positive correlation between each evaluated sperm parameter and TAC levels.BMI and age of participants were not significantly correlated with any sperm parameters (Table 3).

Discussion
The aim of this study was to understand the pathology of nuclear defects leading to poor sperm morphology by analyzing important markers of the OS.In the current study, Sperm DNA/chromatin integrity, OS, and sperm parameters of different infertile men's groups were compared with the normozoospermic men and clarified using standard tests.
In brief, the findings of this study indicated a significant difference between the experimental groups and control group regarding sperm parameters.Sperm chromatin quality and DNA integrity, assessed by TB and CMA3 stains, showed a significant increase in teratozoospermic different groups compared to the control group, indicating defects in protamine and sperm chromatin in infertile men.Our data were in agreement with Ying Ma et al. research suggested that amorphous head familial teratozoospermia might be caused by abnormal chromatin condensation induced by disturbances in the process of histone protamine replacement process, and the elasticity of sperm nuclei could be a new index for assessing the sperm quality [20].Moreover, these findings are in line with the results reported by Ammar et al. who used TUNEL, single-cell gel electrophoresis (comet test), toluidine blue, and acridine orange as four assays to assess the integrity of the nuclear sperm in men with isolated teratozoospermia.The aforementioned experiments demonstrated positive correlations between DNA damage and various morphological anomalies [21,22].In addition, the results of another study by Ammaret al. in 2020 provided clear evidence that apoptotic changes are closely related to abnormal sperm morphology and DNA damage [15].So, sperm protamine deficiency and DNA damage might be one of the pathways which can lead to defects in sperm morphology.OS damage is one of the most important causes of altered sperm morphology and function and is considered an important factor in male infertility [23].Some evidence indicates that the presence of abnormal sperm morphology and motility in semen samples could be due to the OS occurrence in infertile males [24].Furthermore, seminal ROS overproduction may be the primary cause of teratozoospermia [25].
The imbalance between reactive oxygen species and antioxidants causes oxidative stress and may be regarded as an important contributor to infertility in men [26].We measured the level of TOS and TAC in the seminal plasma samples.As expected, the findings of this study showed an increase in the TOS levels and a significant decrease in seminal plasma antioxidant levels in all experimental groups compared to the control group.We also detected a negative correlation between Sperm parameters (concentration, progressive motility, and normal morphology) and CMA3, TB, and TOS levels.There were significant positive correlations between each of the evaluated sperm parameters and TAC levels.In this regard, the results of a study that compared patients with teratozoospermia with fertile men found that the semen ROS production, hypocondensed chromatin, denatured DNA, and fragmented DNA were significantly higher than those for fertile men according to their ROS levels.

Table 2 Comparison of sperm DNA/chromatin integrity and anti-/oxidation status among different groups
Data were not normally distributed (nonparametric) according to the Shapiro-Wilk test.Data were presented as Mean ± SEM.The Kruskal-Wallis H test was used to determine if there are statistically significant differences between all groups and Mann-Whitney test was used to compare between two groups.Bold indicated that P < 0.05 was regarded as a significant value  The semen ROS production, hypocondensed chromatin, denatured DNA, and fragmented DNA were significantly higher than those for fertile men based on their ROS level.The abnormal sperm morphology was correlated positively with all these parameters.A correlation between ROS production and DNA integrity markers was also found.SDF is the main cause of sperm morphology defects and oxidative stress is the major cause of DNA fragmentation in spermatozoa [27].Previous studies also suggested that damage to spermatozoa DNA caused by oxidative stress could have a critical effect on the etiology of infertility [28].Due to oxidative damage to the DNA backbone, poor semen morphology appears to be caused most commonly by defective chromatin compaction, which may induce DNA breaks and free radicals.The DNA backbone may be broken indirectly by reducing protamination and disulfide bond formation due to these [21].Colagar et al. reported that seminal TAC decreased in asthenoteratozoospermia and Oligoasthenoteratozoospermic men compared to healthy individuals, similar to our results [29].In addition, Dehghanpour et al., aiming to compare semen parameters, protamine deficiency, and apoptosis between patients with teratozoospermia (tapered heads) and those with normozoospermia, suggested that apoptosis and abnormal chromatin packaging in tapered-head spermatozoa may contribute to the impaired fertility of teratozoospermic patients with this kind of abnormality, causing impaired fertility in these patients [30].
Treating the patient with oral antioxidant vitamins is often a standard procedure to reduce ROS formation and emend male fertility competency [31].Evidence suggests indiscriminate consumption of antioxidants may damage sperm cells through a reductive-stress-induced state.Therefore, the "antioxidant paradox" must be carefully avoided and fully investigated.Because of these issues, oxidation-reduction potential (ORP) evolved as a valuable tool for assessing the overall balance between oxidants and antioxidants (reductants) in semen [26].
To avoid reductive stress, the oxidative status of the seminal plasma must also be evaluated pre-treatment to proceed with micronutrient supplementation safely.Supplements have the ability to improve male fertility potential and support sperm quality under these conditions [32].
According to recent reports as well as our findings, it seems that evaluating the oxidative status, antioxidant defense systems, and DNA damage, along with semen parameters might be a useful tool for the diagnosing and treating male infertility.More investigation is necessary to improve therapy options and our knowledge of these pathways.

Conclusion
In conclusion, our study demonstrated that DNA defects are much higher in abnormal semen samples than in normozoospermic ones.Also, the subfertile men with poor semen parameters represented significantly higher rates of OSI in comparison with semen samples with normal qualities.From this perspective, simultaneous assessment of sperm DNA defects and OS offers supplementary metrics for sperm quality analysis and may help in determining the most effective treatment method for teratozoospermic men.

Table 1
Comparison of patients' characteristics, semen analysis results between the groupsData were not normally distributed (nonparametric) according to the Shapiro-Wilk test.Data were presented as Mean ± SD.Bold indicated that P < 0.05 was regarded as a significant value.The Kruskal-Wallis H test was used.Experimental group I: Teratozoospermia, Experimental group II: Astheno-teratozoospermia, Experimental group III: Oligoasthenoteratozoospermia, and the control group: Normozoospermia Chromomycin A3; TB Toluidine blue; TAC Total antioxidant capacity; TOS Total oxidant status; OSI Oxidative stress index a Experimental group I: Teratozoospermia b Experimental group II: Astheno-teratozoospermia c Experimental group III: Oligoasthenoteratozoospermia d Experimental group IV: Azoospermia e The control group: Normozoospermia

Table 3
Correlations between sperm parameters and the indicators of chromatin integrity and anti-/oxidation status