The influence of body mass index on oxidative stress markers in infertile men’s semen parameters
Article information
Abstract
Objective
Although various studies have linked environmental toxins, mumps infections, alcohol consumption, and abnormal body mass index (BMI) to impaired semen quality, the precise causes of infertility remain unclear. This study investigates the impact of BMI on oxidative stress markers in semen analysis among infertile men, illuminating the role of oxidative stress in cases of unexplained infertility.
Methods
In this cross-sectional study, 280 patients exhibiting infertility symptoms were recruited. Comprehensive semen analysis was conducted, evaluating reactive oxygen species (ROS) levels, sperm plasma membrane lipid peroxidation via flow cytometry, total antioxidant capacity (TAC), and results from the sperm chromatin structure assay. Participants were categorized based on their BMI, facilitating comparisons between obese and non-obese individuals.
Results
Both BMI and age significantly influenced male fertility, particularly in obese individuals. Strong correlations were identified between elevated BMI, increased ROS levels, and decreased TAC. The obese infertile group exhibited substantially lower TAC compared to controls, highlighting obesity's detrimental effect on antioxidant defenses. Furthermore, significant reductions in sperm count, motility, and normal morphology were observed, alongside an increase in non-motile sperm. These outcomes demonstrate the complex relationship among oxidative stress, BMI, and fertility, emphasizing the necessity for targeted interventions addressing obesity's effects on reproductive health.
Conclusion
This study underscores the importance of managing obesity and understanding its impact on oxidative stress as essential components in improving reproductive outcomes among affected men.
Introduction
Infertility represents a significant global health challenge, affecting approximately 10% to 15% of couples worldwide [1,2]. Male factors contribute to nearly 40% of infertility cases, primarily due to impaired semen quality [3]. While various risk factors, including environmental pollutants, mumps virus infection, and excessive alcohol consumption, have been implicated in deteriorating semen quality, the underlying etiology often remains unclear [4]. Recent investigations have increasingly focused on the role of abnormal body mass index (BMI) in male reproductive health, though findings remain inconsistent. Li et al. [5] demonstrated that elevated BMI is associated with decreased sperm count and concentration in a northern Chinese population. In contrast, Ma et al. [6] reported that both underweight and overweight statuses were linked to reduced total motile sperm counts, suggesting a U-shaped relationship between BMI and semen parameters. Further studies, such as those by Lu et al. [7], suggest that while BMI, waist-hip ratio, and waist circumference (WC) do not directly predict semen quality, significant associations exist between semen quality and hormone levels like follicular stimulating hormone and luteinizing hormone. According to the World Health Organization (WHO), being overweight is defined as having a BMI of 25 kg/m2 or higher, and obesity as a BMI of 30 kg/m2 or more [8]. BMI classifications vary by ethnicity, with obesity defined as BMI ≥27.5 kg/m2 in Asian populations [9]. In Iran, the prevalence of overweight/obesity reaches 59.3% [10]. While most studies report reduced sperm counts in overweight/obese men [11] and negative impacts on sperm motility, morphology, and testosterone levels [12], some studies have found no significant associations [13]. The primary mechanisms involve testosterone suppression and declines in semen quality [14], with emerging evidence implicating oxidative stress [15]. Importantly, weight loss has been shown to improve hormonal profiles, erectile function, and semen quality, highlighting clinical intervention potential [16]. Maintaining a balanced redox state is essential for supporting key sperm functions [17,18]. An imbalance in the production and elimination of reactive oxygen species (ROS) can lead to oxidative damage, adversely affecting sperm quality [18]. Spermatozoa are particularly susceptible to oxidative damage owing to their limited antioxidant defenses and the abundance of polyunsaturated fatty acids in their cell membranes [19]. Under specific pathological conditions, ROS can evolve into highly reactive species that disrupt cellular signaling pathways and inflict considerable damage on critical biomolecules, including nucleic acids, proteins, and lipids [20]. This damaging cascade can compromise membrane integrity, induce mitochondrial dysfunction, reduce sperm motility, and result in DNA damage and programmed cell death (apoptosis) [21]. Obesity has the potential to induce systemic oxidative stress in the testes and sperm, compromising testosterone synthesis, spermatogenesis, and overall sperm quality [22]. Furthermore, increased adipose tissue levels in the scrotal region have shown a correlation with elevated oxidative stress [23]. This oxidative stress arises from an imbalance between ROS production and the activity of three key anti-ROS enzymes: superoxide dismutase, catalase, and glutathione peroxidase. Excessively elevated ROS levels result in cellular damage [24]. While mechanisms underlying oxidative stress in semen and its impact on male reproductive function are increasingly recognized, significant gaps in our understanding persist, warranting further investigation. Therefore, the relationship among ROS, oxidative stress, and fertility/subfertility necessitates continued research. This study aimed to assess the influence of BMI on oxidative stress markers within semen parameters of infertile men.
Methods
1. Sample preparation
This cross-sectional study was conducted at Royan Institute University Hospital, analyzing data from male infertility patients treated between April 2017 and March 2019 (n=280). Although data collection preceded the WHO 2021 semen analysis guidelines, all laboratory procedures were performed using standardized protocols that remain clinically relevant, as validated by recent studies [7]. Participants were categorized into three BMI groups according to current WHO classifications: normal weight (BMI 18.5–24.9 kg/m2, n=75), overweight (BMI 25–29.9 kg/m2, n=75), and obese (BMI ≥30 kg/m2, n=75). The BMI was then calculated using the formula:
All measurements adhered strictly to WHO guidelines and were obtained during the same visit as semen collection. Anthropometric data, specifically each participant’s weight, were recorded using a digital scale with a maximum load capacity of 180 kg. Informed consent was obtained in writing from all subjects, and the study was approved by the Research Ethics Committee of the Royan Institute (IR.ACECR.ROYAN.REC.1401). Participants completed a questionnaire covering demographic information (ethnicity, lifestyle, abstinence period, occupation, and education level), medical history (including reproductive history and genetic risk factors), and lifestyle habits (alcohol and tobacco use). Men with chronic illnesses (diabetes, kidney disease, liver disease, epilepsy, inflammatory bowel disease, atherosclerosis, vascular disease, hypertension, genital disorders, testicular torsion, and genitourinary anomalies), prior testicular surgery, azoospermia, leukocytospermia, or those using medications affecting fertility (including steroids) were excluded. Participant evaluations also considered smoking, alcohol consumption, chemical exposure, hookah usage, testicular surgeries, and duration of infertility. Fertility assessments complied with the latest WHO 2020 guidelines.
1) Semen analysis
Semen samples were collected via masturbation after 2 to 5 days of abstinence and analyzed within 1 hour using WHO 2010 guidelines (which were current during the 2017 to 2019 data collection period). Analysis employed both computer-assisted sperm analysis (SCA v6.4) and manual methods (Neubauer Chamber, Papanicolaou-stained morphology assessments). Although WHO 2021 guidelines were published subsequently, our laboratory’s standardized protocols demonstrated greater than 90% concordance with current criteria for key parameters [7]. Daily calibrations, strict adherence to Kruger morphology criteria, and maintenance of conditions at 37±0.5 °C ensured methodological consistency. Retrospective verification against the 2021 standards confirmed comparable clinical interpretations, validating our approach's ongoing relevance for fertility assessments [25].
2) Measurement of ROS
ROS levels in human semen were quantified using chemiluminescence-based luminol oxidation. Negative controls containing phosphate-buffered saline and positive controls containing H₂O₂ were employed to validate results. Data were reported as relative light units per second per 106 sperm (RLU/sec/106 sperm) after adjusting for sperm concentration [26].
3) Sperm plasma membrane lipid peroxidation
Lipid peroxidation (LPO) in sperm membranes was quantified using a modified thiobarbituric acid (TBA) method (Buege and Aust). A sperm sample and reagent mixture (1:2 ratio) containing trichloroacetic acid, TBA, and hydrochloric acid was heated, cooled, and centrifuged. Malondialdehyde (MDA) concentration in the supernatant was measured spectrophotometrically at 535 nm using an absorbance coefficient of 1.56×105 M-1cm-1. Results were expressed as nmol MDA per 106 spermatozoa [27].
4) Total antioxidant capacity measurement
The total antioxidant capacity (TAC) of semen was determined using a colorimetric assay with a commercial kit from MBL, Germany. Absorbance was measured using a Synergy H4 Hybrid Multi-Mode Microplate Reader by BioTek, and results were expressed as nanomoles per microliter of semen. Sample preparation involved thawing frozen seminal plasma in a 37 °C water bath for 20 minutes, followed by immediate testing per kit instructions [25].
5) Sperm chromatin structure assay
Sperm chromatin integrity was assessed using sperm chromatin structure assay via flow cytometry. Briefly, samples were acid-treated, stained with acridine orange, and analyzed using flow cytometry (10,000 events at approximately 200 cells/second). The DNA fragmentation index (DFI), derived from the ratio of red fluorescence to total fluorescence, was analyzed using FlowJo software (https://www.flowjo.com) [25].
2. Statistical analysis
Statistical analyses were conducted using SPSS software ver. 22 (IBM Co.). Group comparisons utilized either t-tests or Mann-Whitney U tests based on data characteristics. Relationships among variables were evaluated using Pearson’s correlation coefficient. Statistical significance was established at p<0.05.
Results
1. Demographic characteristics of the participants
The demographic characteristics of the study participants are presented in Table 1. Statistically significant differences were identified among the groups for the variables of age, BMI (p<0.001), and duration of infertility (p<0.05). In contrast, variables related to smoking, alcohol consumption, and surgical history were not significantly different among the groups (p>0.05). These results suggest that age and BMI may play a more critical role in differentiating the groups compared to other evaluated variables.
Demographic characteristics
2. Semen analysis results in infertile groups
Our findings revealed significant reductions in sperm count (p=0.016) and total sperm count (p=0.021) in the obese infertile group compared to controls and overweight infertile men (p<0.05). Sperm motility parameters exhibited pronounced decreases in the obese group versus controls: total motility (p<0.001), slowly progressive sperm (p<0.001), A+B progressive sperm (p=0.001), and normal morphology (p<0.001). Although these parameters were reduced relative to controls, they demonstrated a modest but statistically significant increase compared to the overweight infertile group (mean difference 8.3%, p=0.028)—a finding explored further in the ‘Discussion’ regarding possible threshold effects of BMI on spermatogenesis. Conversely, non-motile sperm (p=0.009) and the teratozoospermia index (p<0.001) were elevated in obese participants compared to both control and overweight groups. No significant differences were observed in non-progressive or rapidly progressive sperm motility (p>0.05) (Table 2).
Semen analysis results
3. Assessment of oxidative stress indicators in infertile groups
Table 3 presents the oxidative stress marker analysis across infertile groups. A significant reduction in TAC was observed only in the obese infertile group compared to both control and overweight infertile groups (p=0.000). No significant differences were detected in ROS, LPO, or DFI levels among the groups. These findings indicate that although TAC is notably impaired in obese infertile individuals, other oxidative stress markers may not significantly differ across BMI categories.
Oxidative stress indicators analysis results
4. Analysis of the relationship between sperm parameters and oxidative stress indices
Standardized regression coefficients demonstrating the direction and strength of relationships between sperm parameters and antioxidant indices are shown in Table 4. Among the analyzed variables, BMI and semen volume exhibited significant correlations with ROS levels. Additionally, BMI significantly correlated with TAC, whereas LPO was significantly associated with normal morphology and defects in the sperm neck and mid-piece. The DFI significantly correlated with slowly progressive sperm (class B) and the total number of abnormal sperm. These findings underscore the complex interplay between obesity-related parameters, oxidative stress markers, and sperm quality.
Relationships between sperm parameters and oxidative stress indices
Discussion
Obesity has emerged as a major global health concern, not only due to its established links with metabolic and chronic diseases [28] but also due to its increasingly recognized impact on reproductive health. A growing body of evidence highlights obesity's negative effects on male fertility, including hormonal disruptions, impaired semen parameters, and compromised reproductive function [29]. The demographic findings from our study reinforce these observations, revealing statistically significant differences in age, BMI (p<0.001), and infertility duration (p<0.05) across the groups. These results are consistent with those of Kozopas et al. [30], who identified increased age and elevated BMI as crucial predictors of diminished semen quality and fertility potential in men. These findings are consistent with those reported by MacDonald et al. [31], who identified BMI as a significant predictor of infertility outcomes, underscoring the pivotal roles of age and BMI in differentiating among infertile populations. In contrast, variables such as smoking, alcohol consumption, and surgical history did not show statistically significant differences (p>0.05), consistent with research by Agarwal et al. [32]. Their study suggested that lifestyle factors, although influential, might be overshadowed by other demographic variables such as age and BMI. The observed differences in age among groups—particularly the younger age of men in the normal infertile group (33.1±0.5 years) compared to overweight (36.8±0.7 years) and obese groups (36.1±0.6 years)—have implications for interpreting our results. Although male fertility declines less dramatically with age compared to females, advanced paternal age is associated with reduced semen quality, increased DNA fragmentation, and poorer reproductive outcomes. The younger age of the normal-BMI group may partially explain their shorter duration of infertility (4.04±3.3 months vs. 4.9–5.2 months in other groups), as younger men might seek treatment earlier or present milder underlying conditions. However, significant differences in BMI across the groups remain the primary source of variation.
Our findings revealed significant reductions in sperm count (p=0.016) and total sperm count (p=0.021) among the infertile obese group compared to controls and infertile overweight individuals. These distinctions highlight quantitative impairments in sperm production in the obese population and suggest potential underlying mechanisms. The association between obesity and hormonal alterations is well-documented, with increased adiposity linked to reductions in testosterone and elevated estrogen levels, adversely affecting spermatogenesis [33]. Our results are consistent with previous research indicating obesity's detrimental effects on male reproductive health. Palmer et al. [34], for example, found that obesity negatively impacts spermatogenesis, leading to decreased sperm production. Similarly, Donertas et al. [35] reported significant associations between elevated BMI and impaired sperm parameters, notably sperm concentration and motility. Additionally, some studies have argued that total sperm count might have greater clinical relevance than sperm concentration alone [36].
The observed decline in sperm count among obese infertile men was further exacerbated by significant impairments in sperm motility and morphology. Specifically, parameters such as slowly progressive sperm, A+B progressive sperm, and normal sperm morphology were notably reduced compared to controls. These findings align with Wang et al. [37], who reported similar declines in motility and morphological integrity among obese men. Furthermore, a comprehensive review by Ribeiro et al. [38] underscored the impact of obesity-related factors, such as oxidative stress and hormonal imbalances, on sperm function. Their meta-analysis reinforced the association between increased BMI and compromised sperm motility, emphasizing the need to address obesity as a modifiable risk factor for male infertility [38].
Conversely, we observed a significant increase in non-motile sperm and elevated teratozoospermia indices within the infertile obese group compared to both control and overweight groups. The elevated teratozoospermia index, indicating a higher proportion of morphologically abnormal sperm, raises concerns about sperm viability in this subgroup [39]. Teratozoospermia is linked to reduced fertility potential, and its increased prevalence in obese individuals indicates significant reproductive challenges [40]. These findings suggest the importance of adopting a multifaceted approach to male infertility management, emphasizing lifestyle modifications alongside an improved understanding of the complex relationship between BMI and reproductive health [40]. The significant reduction in TAC observed in obese infertile individuals (p=0.00) highlights obesity’s adverse effects on antioxidant defense mechanisms. This result aligns with earlier findings indicating that obesity promotes oxidative stress, severely compromising the body's capacity to neutralize free radicals. A study by Guan et al. [41] reported similarly diminished TAC levels among obese individuals, indicating increased susceptibility to oxidative damage. Additionally, Chrysohoou et al. [42] identified an inverse relationship between visceral fat and TAC, a correlation notably stronger when WC was considered rather than BMI alone. Conversely, our study did not detect significant differences in other oxidative stress markers such as ROS, LPO, and DFI. This contrasts with Jing et al. [43], who found that obesity generally elevates oxidative stress markers. This variance may suggest a complex interplay in which TAC and specific oxidative markers do not respond uniformly to obesity, emphasizing the need for more nuanced research [43]. Our analysis also revealed significant correlations between BMI, semen volume, and ROS levels, highlighting obesity's role in promoting oxidative stress and subsequent sperm dysfunction [44]. Elevated BMI was notably associated with reduced TAC, indicating impaired antioxidant defenses in obese individuals. Furthermore, increased LPO correlated significantly with sperm abnormalities, particularly impacting normal morphology and defects in the sperm neck and mid-piece regions. These findings collectively indicate that obesity-induced oxidative stress, characterized by both increased ROS production and reduced antioxidant capacity, significantly impairs sperm structure and function, ultimately compromising fertility potential [45]. Lastly, variations in the DFI exhibited significant relationships with slowly progressive sperm (class B) and the total number of abnormal sperm. Higher oxidative stress levels likely contribute to increased DNA fragmentation, severely impairing sperm viability and fertilization potential [46]. Together, these findings emphasize the intricate connections among obesity-related parameters, oxidative stress markers, and sperm quality, highlighting their critical implications for male fertility.
This study demonstrates significant associations between elevated BMI and oxidative stress markers, particularly reflected in reduced TAC and increased ROS production. The obese infertile group exhibited notably lower TAC levels compared to both control and overweight infertile groups, indicating substantial impairment of antioxidant defenses due to obesity. These metabolic alterations corresponded with clinically relevant declines in semen quality parameters, including reduced sperm count, diminished motility, impaired morphology, and elevated teratozoospermia indices, collectively indicating a direct detrimental effect of obesity on spermatogenesis. Notably, while LPO and DFI did not show significant variations, the pronounced reduction in TAC reveals a selective yet impactful oxidative stress pathway in obese individuals. These findings confirm obesity as a key modifiable risk factor for male infertility and highlight the necessity of: (1) mechanistic studies to elucidate specific oxidative pathways; (2) clinical interventions targeting metabolic improvement in obese men seeking fertility treatment.
The consistent deterioration observed across multiple semen parameters strongly suggests that obesity-mediated oxidative stress significantly contributes to subfertility, warranting further clinical attention and investigation.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
We would like to thank the Royan Institute clinic for implementing in this plan.
Author contributions
Conceptualization: RH, MS. Methodology: ZM, SA. Formal analysis: ZM. Data curation: MMS. Project administration: MS. Visualization: MMS. Validation: SA. Investigation: ZM. Writing-original draft: ZM. Writing-review & editing: SA, MS. Approval of final manuscript: ZM, SA, MMS, RH, MS.