Introduction
Reproduction is a complex physiological process crucial for the continuation of a species. In humans, it involves the fusion of male and female gametes in humans. Beyond its biological necessity, reproduction also reflects a deep, personal desire for parenthood.
However, achieving pregnancy is not always straightforward, and more than half of couples experiencing infertility will require medical intervention to address the underlying issues [1]. Infertility affects approximately 80 million couples worldwide [2], with male infertility contributing to up to 50% of cases [3].
In 2009, the World Health Organization (WHO) defined infertility as a disease of the reproductive system, characterized by the inability to achieve a clinical pregnancy after 12 months of regular, unprotected intercourse [4]. Infertility can stem from a variety of causes, including male and female factors, or a combination of both [5].
Oxidative stress, a significant contributor to infertility, was first defined by Sies [6] in 1991. It describes a condition characterized by an imbalance between pro-oxidants and antioxidants, resulting in cellular damage when free radicals surpass the body's ability to neutralize them [6].
Oxidative stress damages cellular components, particularly DNA, lipids, and proteins [7]. It is associated with a broad spectrum of health issues, including cancers, cardiovascular diseases, neurological disorders, and infertility [8-10].
Free radicals can originate from both endogenous and exogenous sources. Endogenous sources encompass metabolic processes in mitochondria and the endoplasmic reticulum, as well as immune responses such as phagocytosis and inflammation. Exogenous sources include environmental and lifestyle factors like smoking, alcohol consumption, poor diet, stress, and exposure to pollutants, heavy metals, and radiation [11,12].
A significant consequence of oxidative stress is lipid peroxidation. This process occurs when free radicals attack polyunsaturated fatty acids within cell membranes, leading to the formation of malondialdehyde (MDA), which serves as a marker of cellular damage [13].
To counteract oxidative stress, the body employs several crucial antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). These enzymes help neutralize harmful molecules like hydrogen peroxide and superoxide radicals, aiding in the restoration of redox balance and protecting cells from oxidative damage [14]. Maintaining a delicate balance between lipid peroxidation and the activity of these antioxidant enzymes is essential for preserving homeostasis and preventing a range of diseases associated with oxidative damage.
This study aimed to assess lipid peroxidation (MDA levels) and antioxidant enzyme activities (CAT, SOD, and GPx) in seminal fluid. By examining the relationship between oxidative stress and sperm health, we seek to deepen our understanding and potentially improve male fertility.
Methods
1. Design and settings
A prospective, descriptive cohort study was conducted from January 2021 to January 2023 at the Integrated Physiology Laboratory (LR17ES02), Faculty of Sciences, Bizerte, University of Carthage, Tunisia. This study was carried out in collaboration with the Alyssa Fertility Group at the Alyssa Clinic, located in Les Berges du Lac, Tunis.
2. Ethics
The study adhered to the ethical standards set by national and institutional committees on human experimentation and the Helsinki Declaration (1975, revised 2008) [15]. It was approved by the Alyssa Clinic Ethics Committee (Ref: AFG/MBR/11/20), and informed consent was obtained from all participants.
3. Participants and study groups
The participants were selected based on their infertility assessments and/or eligibility for an intracytoplasmic sperm injection attempt, having experienced infertility for more than 1 year. The study primarily concentrated on male infertility, excluding any cases of female infertility.
A total of 163 patients were classified into five groups based on their semen parameters: normozoospermia with a sperm count ≥15 million/mL (n=39), oligozoospermia with a sperm count <15 million/mL (n=36), asthenozoospermia with less than 42% motile sperm (n=33), teratozoospermia with more than 4% sperm exhibiting normal morphology (n=28), and oligo-astheno-teratozoospermia, which combines the three aforementioned parameters (n=27). For the normozoospermia group, we selected male volunteers who had fathered children within the year preceding the study. This normozoospermic group served as the control group.
4. Exclusion criteria
Exclusion criteria included men with a history of serious illnesses, those who had taken medications or undergone antioxidant treatments within the past 3 months, and patients diagnosed with azoospermia, urogenital infections, or febrile conditions.
5. Sample collection and semen analysis
Sperm analyses were performed according to the WHO guidelines [16,17]. The sperm count was determined using a semi-automated method with the Sperm-Class-Analyzer software (SCA5/6 computer-assisted sperm analysis) and SCA-scope (Microptic). All patients underwent a comprehensive assessment of their sperm characteristics, with one or two sperm analyses performed following a period of sexual abstinence lasting 2 to 3 days.
For sperm motility, following the WHO recommendations [17], we used a classification system divided into four grades: grade a: rapid progressive motility in a straight line (speed >25 µm/sec); grade b: slow progressive motility (speed of 5–25 µm/sec); grade c: non-progressive motility or slight movement; and grade d: immotile sperm.
Sperm morphology was analyzed descriptively, classifying various anomalies in the spermatozoa's head, midpiece, and flagellum. Head abnormalities were categorized as either microcephalic or macrocephalic. Midpiece defects included asymmetry, thickening, bulging, irregularity, and the presence of residual cytoplasm. Flagellar abnormalities were classified as short, angular, coiled, double, or multiple flagella, as well as instances of flagellum-free spermatozoa.
6. Oxidative stress measurement
1) Malondialdehyde assay
Lipid peroxidation was assessed in spermatozoa-free seminal plasma using the thiobarbituric acid (TBA) reacting substance (TBARS) method to specifically quantify MDA levels [18]. Semen samples were initially centrifuged for 10 minutes at 1,800 ×g. Subsequently, 500 µL of the supernatant (seminal fluid) was mixed with 10% trichloroacetic acid (TCA) and 0.67 g of TBA. This mixture was then heated in a boiling water bath for 30 minutes. After heating, an equal volume of n-butanol was added. The mixture was centrifuged again, and the organic phase was collected for fluorescence measurements. To prevent artefactual lipid peroxidation during the boiling step, 1 mM butylated hydroxytoluene was added to the samples. The absorbance of the samples was measured at 532 nm, and the results were expressed as nanomoles of MDA per gram of protein.
2) Catalase activity
CAT activity was measured according to the method described by Aebi [19]. The activity was assessed by monitoring the rate of hydrogen peroxide degradation at 240 nm in a 10 mM potassium phosphate buffer with a pH of 7.0. An extinction coefficient of 0.036 mM/cm was utilized for the calculations. One unit of activity was defined as the consumption of 1 pmol of hydrogen peroxide per minute, and the specific activity was expressed as units per milligram of protein.
3) Activity of superoxide anion production
The specific activity of SOD was measured using a slightly modified version of the method described by Misra and Fridovich [20]. Briefly, 485 μL of NaHCO3–Na2CO3 buffer (pH 10.2, 10⁻⁴ M ethylenediamine tetraacetic acid) is added, followed by 15 μL of epinephrine (10⁻² M in HCl solution) at 30 °C. The absorbance change at 480 nm was recorded, and the rate of change in optical density (ΔOD/min) was calculated. The same procedure was repeated with a 25-μL aliquot of seminal fluid. The ΔOD/min of the sample was compared to that of a standard epinephrine solution.
4) Glutathione peroxidase
GPx activity was determined using the method by Flohe and Gunzler [21]. A 200 μL aliquot of seminal fluid was mixed with 100 μL of phosphate-buffered saline (0.1 M; pH=7.4) and 200 μL of glutathione (GSH; 4 mM). This mixture was incubated in a water bath at 37 °C for 10 minutes. After 1 minute, 1 mL of 5% TCA was added to 0.5 mL of 5 mM H₂O₂. After centrifugation, the supernatant was collected. To this, 2 mL of phosphate buffer and 0.4 mL of 10 mM 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) were added. The reaction was measured at 420 nm.
7. Statistical analysis
Statistical analysis was performed using SPSS ver. 25.0 (IBM Co.). Data normality was assessed with the Kolmogorov–Smirnov test. Group comparisons were made using analysis of variance with the Tukey post hoc test. Pearson correlation analysis was applied based on the data distribution. A p-value of <0.05 was considered statistically significant.
Results
1. Data summary of male patients by semen quality
The study evaluated semen quality in five groups of male patients: normozoospermia (39 patients), oligozoospermia (36 patients), asthenozoospermia (33 patients), teratozoospermia (28 patients), and oligo-astheno-teratozoospermia (27 patients).
All patients underwent one or two detailed sperm analyses. The period of sexual abstinence prior to the day of analysis was consistent across all groups, averaging between 3.10 and 3.28 days. The patients' ages were comparable, ranging from 36.4 to 37.5 years. Ejaculate volume was relatively consistent, with normozoospermia averaging 3.08 mL, and volumes in other groups varying from 2.91 to 3.12 mL. Leukocyte counts were also similar across all groups, ranging from 0.49 to 0.52 ×106/mL, indicating no significant inflammatory response (Table 1).
A significant decrease in sperm count was observed in the pathological groups. In normozoospermia, the average sperm concentration was 88.15 million/mL. In contrast, concentrations in oligozoospermia, asthenozoospermia, teratozoospermia, and oligo-astheno-teratozoospermia were 7.18, 23.03, 17.85, and 2.31 million/mL, respectively. Additionally, the percentage of sperm with abnormal morphology increased as semen quality declined. The normozoospermic group exhibited an abnormal morphology rate of 87.65%, whereas the teratozoospermic group showed a significantly higher rate, reaching 98.16%. Progressive motility (a+b) also significantly declined in groups with compromised semen quality. For instance, normozoospermia demonstrated a progressive motility rate of 58.31%, while oligo-astheno-teratozoospermia had a markedly lower rate of 11.51% (Table 1).
2. Effects on lipid peroxidation
Our results showed a significant increase (p<0.05) in MDA levels in all pathological groups compared to the control group, which had an MDA value of 0.354±0.208 nmol/mg protein. MDA levels were significantly elevated across the study groups: the oligozoospermia group exhibited levels of 1.873±0.284 nmol/mg protein, the asthenozoospermia group had an average of 2.197±0.292 nmol/mg protein, and the teratozoospermia group recorded 2.264±0.225 nmol/mg protein. The highest MDA level was observed in the oligo-astheno-teratozoospermia group, reaching 2.316±0.418 nmol/mg protein (Figure 1A).
3. Effects on catalase
A significant reduction in CAT activity was observed in the seminal fluid across all pathologic groups studied (p<0.05). In the control group, CAT activity was measured at 4.392±0.492 µmol H2O2/min/mg protein. This value decreased by 76% in the oligozoospermia group to 1.014±0.492 µmol H2O2/min/mg protein; by 63% in the asthenozoospermia group to 1.619±0.921 µmol H2O2/min/mg protein; by 94% in the teratozoospermia group to 0.254±0.165 µmol H2O2/min/mg protein; and by 95% in the oligo-astheno-teratozoospermia group to 0.186±0.072 µmol H2O2/min/mg protein (Figure 1B).
4. Effects on superoxide dismutase
SOD activity exhibited a significant decrease (p<0.05) across all pathological groups in the seminal fluid. In the control group, SOD activity was measured at 21.86±3.22 µmol/mg protein. The oligozoospermia group showed a decrease to 14.64±2.87 µmol/mg protein, reflecting a 33% reduction. SOD activity in the asthenozoospermia group was 12.75±2.43 µmol/mg protein, indicating a 41% reduction. The teratozoospermia group recorded an SOD level of 12.96±2.90 µmol/mg protein, representing a 40% reduction. The lowest SOD activity was observed in the oligo-astheno-teratozoospermia group, at 9.22±1.60 µmol/mg protein, corresponding to a 57% decline (Figure 1C).
5. Effects on glutathione peroxidase
GPx activity significantly decreased (p<0.05) across all pathological groups. The control group exhibited a GPx activity of 96.74±10.29 µmol of reduced GSH/mg protein. In the oligozoospermia group, GPx activity decreased by 18% to 78.85±4.46 µmol of reduced GSH/mg protein. The asthenozoospermia group experienced a 33% reduction, with GPx activity at 64.76±5.99 µmol of reduced GSH/mg protein. In the teratozoospermia group, activity decreased by 30% to 67.00±6.26 µmol of reduced GSH/mg protein, and the oligo-astheno-teratozoospermia group exhibited a 35% decline, recording 61.92±5.90 µmol of reduced GSH/mg protein (Figure 1D).
6. Correlations between oxidative stress markers and sperm parameters
Correlations between various oxidative stress markers (MDA, CAT, SOD, and GPx) and sperm parameters were analyzed in individuals with normal sperm quality and various forms of male infertility (Table 2). In groups with lower sperm quality, significant negative correlations were observed between sperm concentration and MDA (r=–0.881, p<0.001), indicating that oxidative stress may reduce sperm concentration. The antioxidant enzyme CAT demonstrated significant positive correlations with sperm concentration in the oligozoospermia, asthenozoospermia, and teratozoospermia groups. However, correlations between CAT and GPx were negative in the oligo-astheno-teratozoospermia group.
MDA exhibited a negative correlation with motility in cases of asthenozoospermia (r=–0.863, p<0.001), teratozoospermia (r=0.710, p<0.001), and oligo-astheno-teratozoospermia (SOD: r=–0.752, p<0.01). This suggests that oxidative stress impairs motility. Conversely, antioxidant enzymes (CAT, SOD, and GPx) showed a positive correlation with sperm motility in these groups, indicating that antioxidant activity enhances sperm motility.
MDA demonstrated a positive correlation with abnormal morphology in cases of oligozoospermia and asthenozoospermia, suggesting that oxidative stress is associated with increased morphological abnormalities. Conversely, antioxidants (CAT, SOD, and GPx) showed negative correlations with abnormal morphology, especially in teratozoospermia (CAT: r=–0.864, p<0.01; SOD: r=–0.885, p<0.01) and oligo-astheno-teratozoospermia, indicating that antioxidants may help preserve sperm morphology (Table 2).
In summary, oxidative stress, as indicated by MDA levels, is associated with reductions in sperm concentration, motility, and morphology. Conversely, antioxidant enzymes such as CAT, SOD, and GPx seem to safeguard sperm quality, especially in groups exhibiting impaired sperm parameters.
Discussion
The analysis of male patients across various groups showed consistent results in terms of sexual abstinence, age, and ejaculate volumes. However, significant declines in sperm count, motility, and morphology were observed under pathological conditions. These findings are consistent with previous research, which indicates that low sperm counts are strongly associated with decreased fertility potential, especially in cases of oligozoospermia, a leading cause of male infertility [22].
While sperm morphology has long been considered a critical factor in fertility, its role remains a subject of debate. Abnormal sperm morphology is associated with lower fertilization rates; however, its overall significance in male fertility is limited when considered alongside other parameters such as sperm motility and count [23]. Our study's findings underscore the critical importance of motility for successful conception. Impaired motility, a key determinant of a sperm's ability to reach the egg, was significantly reduced in all pathological groups, highlighting its negative impact on fertility outcomes [24].
The absence of significant inflammatory responses across the groups indicates that the observed sperm abnormalities are not due to inflammation. This observation aligns with research suggesting that environmental toxins, lifestyle choices, and oxidative stress can impair male fertility [25]. These elements can degrade sperm quality without triggering noticeable inflammation.
Our study on oxidative stress markers (MDA, CAT, SOD, and GPx) is consistent with existing research on male infertility. We observed elevated levels of MDA and decreased activities of antioxidant enzymes, suggesting that oxidative stress significantly contributes to the decline in sperm quality and conditions such as oligozoospermia, asthenozoospermia, teratozoospermia, and oligo-astheno-teratozoospermia. Notably, the highest MDA levels were found in the oligo-astheno-teratozoospermia group.
Sperm is highly vulnerable to oxidative stress due to its rich content of polyunsaturated fatty acids and a lack of cytoplasmic antioxidant enzymes [26]. This susceptibility leads to oxidative damage, which can impair normal fertilization [27]. Elevated levels of MDA are associated with decreased sperm motility and abnormal morphology [28] and contribute to infertility [29]. Our findings corroborate these observations, demonstrating a direct correlation between high MDA levels and poor sperm quality, especially in the oligo-astheno-teratozoospermia group.
Unexpectedly, serum MDA levels were higher in normozoospermic men than in those with other fertility impairments. This finding is surprising because high MDA levels, typically associated with oxidative stress, are generally linked to reduced fertility [30]. These results suggest a more complex and potentially indirect relationship between MDA levels and male fertility, warranting further research to explore the underlying mechanisms.
In addition to MDA, activities of antioxidant enzymes were significantly reduced in men with sperm abnormalities. Our study observed a substantial decrease in CAT activity across all pathological groups. These reductions indicate a compromised antioxidant defense system in men with various forms of infertility. CAT plays a crucial role in converting hydrogen peroxide into water and oxygen, thereby protecting cells from oxidative damage.
Recent studies confirm the association between reduced CAT activity and male infertility, noting that infertile men typically have significantly lower CAT levels, which diminishes their ability to neutralize harmful reactive oxygen species (ROS) such as hydrogen peroxide [30,31]. Similarly, SOD activity in seminal fluid was found to be significantly decreased across all pathological groups, aligning with previous research that associates lower SOD levels with increased oxidative stress and impaired sperm motility [32]. Additionally, sperm from teratozoospermic individuals produce higher levels of ROS compared to morphologically normal sperm, which exacerbates oxidative damage and compromises sperm function [33]. Our results further support the hypothesis that oxidative damage, caused by elevated ROS levels, contributes to sperm dysfunction.
However, Hsieh et al. [34] found no significant correlation between SOD activity in sperm and seminal plasma and sperm quality, suggesting that SOD levels alone may not reliably predict sperm fertilization potential. This raises questions about the utility of measuring SOD as a sole marker of male fertility and highlights the need for a more comprehensive approach to assessing oxidative stress and sperm health.
GPx activity, another crucial antioxidant enzyme, was significantly reduced in all pathological groups. These reductions are consistent with findings by Ribeiro et al. [35], who observed lower GPx levels in severe cases of asthenozoospermia, oligozoospermia, and teratozoospermia. However, some studies indicate that GPx activity may not consistently correlate with semen quality, suggesting variability across different populations or conditions [36].
GPx, an enzyme that contains selenocysteine, plays a crucial role in protecting sperm cells from oxidative damage [37]. A decrease in its activity can compromise sperm integrity, leading to damage and reduced fertility. In line with the findings of Crisol et al. [38], who found a positive correlation between GPx activity and semen quality, our results further emphasize the weakened antioxidant defenses associated with male infertility. This highlights the potential role of oxidative stress in the pathophysiology of impaired fertility.
In conclusion, this study presents compelling evidence linking oxidative stress to poor sperm quality in cases of male infertility, particularly in conditions such as oligozoospermia, asthenozoospermia, and teratozoospermia. The findings indicate that a weakened antioxidant defense system plays a significant role in sperm dysfunction, positioning oxidative stress as a critical factor in male infertility. However, the results also highlight the necessity for a more nuanced, multifactorial approach to assessing oxidative stress in fertility contexts. Further research is essential to investigate targeted antioxidant interventions for managing infertility associated with oxidative damage.
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