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Keywords:

  • mitochondria/metabolism;
  • DNA damage;
  • obesity;
  • sperm

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Study Type – Prognosis (cohort)

Level of Evidence 3a

What's known on the subject? and What does the study add?

The relationship between high levels of BMI and changes in altered standard semen analysis parameters are described in the literature. However, the functional characteristics of the sperm are essential to complete the evaluation of male infertility. Thus, this study provides important information about the functionality of the sperm of men with different levels of BMI.

OBJECTIVE

  • • 
    To assess the effect of obesity on semen analysis, sperm mitochondrial activity and DNA fragmentation.

MATERIALS AND METHODS

  • • 
    A transversal study of 305 male patients, presenting for clinical evaluation, was carried out. The patients were divided into three groups according to body mass index (BMI) as follows: eutrophic (BMI < 25 kg/m2, n= 82), overweight (BMI ≥ 25 kg/m2 and <30, n= 187) and obese (BMI ≥ 30 kg/m2, n= 36).
  • • 
    The variables analysed were semen analysis, rate of sperm DNA fragmentation and sperm mitochondrial activity.
  • • 
    Groups were compared using one-way analysis of variance followed by a least significant difference post-hoc test. A P-value of <0.05 was considered to indicate statistical significance.

RESULTS

  • • 
    No differences were observed in age, ejaculatory abstinence, ejaculate volume, sperm vitality, morphology or round cell and neutrophil count among the groups.
  • • 
    The eutrophic group had a higher percentage of sperm with progressive motility (P= 0.001). Mitochondrial activity was lower in the obese group (P= 0.037) when compared to the eutrophic, and the percentage of sperm with DNA damage was higher in the obese group (P= 0.004) than the other two groups.

CONCLUSION

  • • 
    Increased BMI values are associated with decreased mitochondrial activity and progressive motility and increased DNA fragmentation.

Abbreviations
BMI

body mass index

LSD

least significant difference

ROS

reactive oxygen species

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Infertility, defined as the inability to achieve pregnancy after 12 months of sexual intercourse without the use of contraceptives [1], affects around 15% of couples of reproductive age, and male factor infertility is the cause in up to 50% of these cases [1,2]. Different aetiologies may be associated with male factor infertility, such as varicocele, cryptorchidism, genetic alterations, systemic diseases, hypogonadotropic hypogonadism, and altered semen analysis values [3,4]. Obesity was recently also included in this list [5–7].

The effects of excess body fat in female fertility are well documented [8,9], but results are still scarce and controversial with regard to male fertility [5,10,11]. Obesity can negatively affect fertility through various mechanisms, many not yet clarified. Higher body mass index (BMI) values result in an altered reproductive hormone profile, with lower testosterone and sex hormone-binding globulin levels, higher oestradiol levels and, in extreme cases, changes in gonadotrophin secretion [12]. Levels of inhibin B, a marker of Sertoli cell function and spermatogenesis, may also be altered in these cases [5,13]. In addition, the accumulation of suprapubic and inner thigh fat may lead to an increase in scrotal temperatures (hyperthermia) in obese men. This affects spermatogenesis by causing testicular oxidative stress [14,15].

Most reports describe the relationship between obesity and altered standard semen analysis parameters [5,16,17], but lack information regarding sperm functional analysis. It is important to evaluate the consequences of obesity with regard to sperm function in men. The aim of the present study was to assess the impact of higher BMI values on sperm mitochondrial activity and DNA integrity.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

STUDY POPULATION

A transversal study, involving 305 patients presenting to the Human Reproduction Section of the Sao Paulo Federal University, was carried out. Each patient's percentage of body fat was estimated by calculating their weight/height ratio, i.e. their BMI [18]. BMI was assessed for all patients during a physical examination performed by the same physician. Patients were divided into three groups according to BMI: eutrophic (BMI < 25.0 kg/m2; n= 82), overweight (BMI ≥ 25.0 kg/m2 and <30 kg/m2; n= 187) and obese (BMI ≥ 30.0 kg/m2; n= 36) groups.

Exclusion criteria were current or previous urogenital diseases, current or previous systemic diseases that would lead to testicular alterations, e.g. cancer (and chemotherapy), and endocrinopathies. Institutional Review Board approval was obtained from the Sao Paulo Federal University Research Ethics Committee.

SEMEN ANALYSIS

Semen samples were collected by masturbation after 2–7 days of ejaculatory abstinence. After semen liquefaction, seminal analysis was performed according to WHO criteria [1] and sperm morphology was evaluated using Kruger et al.'s strict criteria [19]. An aliquot was used for evaluation of sperm mitochondrial activity and DNA fragmentation.

EVALUATION OF MITOCHONDRIAL ACTIVITY

To determine sperm mitochondrial activity, the method proposed by Hrudka [20] was used, based on the oxidation, polymerization and deposition of DAB by mitochondrial cytochrome c oxidase. Briefly, semen was diluted 1:1 to 1:3 in a solution containing 1 mg/mL of DAB in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) and incubated at 37 °C for 1 h in the dark. Two 10-µL smears were then prepared on microscope slides and air dried. The slides were fixed in 10% formaldehyde for 10 min, washed and air dried again. A total of 200 sperm were counted using a 1000X magnification differential interference contrast Olympus BX51 microscope, and cells were classified as: class I (100% of the midpiece was stained), class II (>50% of the midpiece was stained), class III (<50% of the midpiece was stained) and class IV (absence of staining in the midpiece).

DETERMINATION OF DNA INTEGRITY

To evaluate sperm nuclear DNA integrity, a modified alkaline single-cell gel electrophoresis, or comet assay, was performed on semen samples from each study subject, as described previously [21]. Slides were pre-coated with 1% normal melting point agarose (GE Healthcare, Amersham, UK) in TBE (0.089M Tris base, 0.089M borate and 0.002M EDTA) overnight. A 100-µL aliquot of fresh semen diluted to a final concentration of 1 × 106/mL in 0.75% low melting point agarose (GE Healthcare) in TBE was added to each slide. This was covered with a coverslip and kept for 10 min at 4oC to solidify. After the gel had solidified, the coverslips were gently removed and 300 µL of 0.75% low melting point agarose in TBE were added. After 10 min, the slides were immersed in cold lysis solution (100 mM Na2-EDTA, 10 mM Tris, 2.5 M NaCl, pH = 11, 4 mM dithiothreitol, 2% Triton X-100) for 2 h.

The slides were then immersed in Milli-Q water (Millipore, Billerica, MA, USA) for 10 min, to remove the excess salts, then immersed in alkaline electrophoresis solution (300 mM NaOH, 1 mM Na2-EDTA, pH > 13) for 20 min. Electrophoresis was performed for 20 min at 3 V/cm, 150–300 mAmp. The slides were then covered with TBE (pH = 7.4) for 15 min and fixed with ethanol 100% (2 × 5 min). After drying, the slides were stained with ethidium bromide (7 µg/mL; Invitrogen, Carlsbad, CA, USA) for 15 min and washed with TBE (3 × 5 min) to remove background staining. The slides were then evaluated using an Olympus BX51 epifluorescence microscope equipped with a rhodamine/TRITC filter and a 100-W mercury lamp. A total of 200 sperm cells were scored according to intensity of DNA damage, as assessed by comet tail and nuclear intensity, and visually classified using a scale of I (high DNA integrity) to IV (high DNA fragmentation). Class I cells presented a nucleus with intense fluorescence and did not present a comet tail. Class II cells still presented an evident nucleus but also a comet tail. Class III cells presented a weak nucleus and a strong tail and Class IV cells did not present a nucleus, only a comet tail.

STATISTICAL ANALYSIS

Statistical analysis was performed using SPSS 13.0 for Windows. All variables were initially tested to determine variance homogeneity and data normality, and heteroscedastic data were transformed. Groups were compared using one-way anova, with a least significant difference (LSD) test for post-hoc comparison. A statistical power of α= 0.005 was adopted. Data are presented as mean (sd) with 95% CIs.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

ROUTINE SEMEN ANALYSIS

No differences were observed in age, ejaculatory abstinence, ejaculate volume, sperm vitality, morphology or round cell and neutrophil counts (P > 0.10). There was a tendency for the eutrophic group to have a higher sperm concentration than the obese group: 80.5 (70.5) × 106/mL vs 48.3 (55.8) × 106/mL (P= 0.08).

Progressive motility significantly differed between the eutrophic group and both the overweight and obese groups (P= 0.001). This difference could also be observed in the total number of motile sperm (P= 0.012). Consequently, the percentage of static sperm was higher in the overweight and obese groups (P= 0.002; Table 1).

Table 1.  Age, ejaculatory abstinence and semen analysis results in men in the different BMI groups
 Eutrophic groupOverweight groupObese group P
  • *

    Significant difference (P < α). Different superscript letters in a same row indicate significant (P < 0.05) differences (LSD post-hoc test).

Age, years     
 Mean (sd)33.5 (6.1)34.7 (7.9)34.3 (4.9)0.462
 95% CI32.2; 34.933.6; 35.932.6; 36.0 
Abstinence, days     
 Mean (sd)4.0 (2.1)4.3 (2.3)4.0 (1.7)0.551
 95% CI3.5; 4.53.9; 4.63.4; 4.5 
Volume, mL     
 Mean (sd)3.2 (1.3)3.1 (1.5)2.8 (1.2)0.357
 95% CI2.9; 3.52.9; 3.32.4; 3.2 
Concentration, ×106/mL     
 Mean (sd)80.5 (70.5)66.8 (75.8)48.3 (55.8)0.078
 95% CI65.0; 96.055.8; 77.729.4; 67.2 
Motility a + b, %     
 Mean (sd)54.8 (13.7)a48.3 (14.9)b45.0 (15.7)b0.001*
 95% CI51.8; 57.846.1; 50.439.6; 50.3 
Motility d, %     
 Mean (sd)40.2 (13.0)a46.4 (15.3)b48.8 (14.7)b0.002*
 95% CI37.4; 43.144.2; 48.643.8; 53.7 
Total motile, ×106     
 Mean (sd)134.7 (134.4)a96.4 (120.3)b68.7 (100.1)b0.012*
 95% CI105.2; 164.379.0; 113.834.9; 102.6 
Morphology, % normal     
 Mean (sd)8.6 (4.2)8.0 (4.5)8.3 (3.7)0.652
 95% CI7.6; 9.57.4; 8.77.0; 9.5 
Round cell, ×106/mL     
 Mean (sd)2.9 (2.9)3.4 (4.0)3.9 (5.0)0.427
 95% CI2.3; 3.62.9; 4.02.2; 5.6 
Neutrophiles, ×106/mL     
 Mean (sd)0.6 (1.2)1.0 (2.7)1.0 (1.6)0.369
 95% CI0.3; 0.90.6; 1.40.5; 1.6 

DETERMINATION OF DNA INTEGRITY AND MITOCHONDRIAL ACTIVITY

The obese group had a lower percentage of sperm with mitochondrial activity DAB class II (P= 0.045) and a higher percentage of sperm with low or zero mitochondrial activity (DAB classes III and IV, P= 0.002 and 0.037, respectively) than the other two groups.

The same was observed for the rate of sperm DNA fragmentation; obese men had a higher percentage of sperm with high DNA fragmentation (comet class IV, P= 0.004 [Table 2]).

Table 2.  Mitochondrial activity and DNA integrity values in men in the different BMI groups
 Eutrophic groupOverweight groupObese group P
  • *

    Significant difference (P < α). Different superscript letters in a same row indicate significant (P < 0.05) differences (LSD post-hoc test).

DAB I (%)     
 Mean(sd)10.0 (9.88.1 (8.1)7.3 (9.2)0.176
 95% CI7.8; 12.16.9; 9.24.2; 10.4 
DAB II (%)     
 Mean(sd)70.8 (11.8)a67.6 (14.3)a,b64.2 (16.7)b0.045*
 95% CI68.3 (73.465.5 (69.658.5 (69.8 
DAB III (%)     
 Mean(sd)11.5 (5.3)a14.1 (6.7)b15.4 (8.5)b0.002*
 95% CI10.3; 12.613.1; 15.112.5; 18.3 
DAB IV (%)     
 Mean(sd)7.7 (8.4)a10.2 (11.6)a,b13.1 (11.7)b0.037*
 95% CI5.8; 9.58.5; 11.99.1; 17.1 
Comet I (%)     
 Mean (sd)42.3 (23.7)41.3 (23.4)37.8 (22.2)0.617
 95% CI37.1; 47.538.0; 44.730.3; 45.3 
Comet II (%)     
 Mean (sd)43.0 (20.0)41.4 (19.2)42.0 (16.8)0.812
 95% CI38.7; 47.438.6; 44.236.3; 47.7 
Comet III (%)     
 Mean (sd)11.4 (7.3)12.5 (7.5)12.5 (8.2)0.525
 95% CI9.8; 13.011.5; 13.69.7; 15.3 
Comet IV (%)     
 Mean (sd)4.4 (5.0)a4.7 (4.6)a7.7 (8.3)b0.004*
 95% CI3.3; 5.54.0; 5.34.9; 10.5 

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Overweight and obesity, defined as abnormal or excessive fat accumulation, are associated with an increased risk of many health problems, including type II diabetes, cardiovascular disease, hypertension, dyslipidaemia, cancers and infertility [22]. The currently high rates of obesity are attributable to the increase in sedentary lifestyles and, in particular, to changes in diet [23].

Recently, the association of BMI with standard semen analysis parameters has been examined in several studies. Sallmen et al.[17] observed that a three-unit increase in male BMI was associated with infertility. In another study, Jensen et al.[5] observed that high (>25 kg/m2) or low (<20 kg/m2) BMI were associated with reduced semen quality (e.g. sperm concentration and total sperm count). Percentages of normal spermatozoa were lower, although not significantly, among men with high or low BMI. Semen volume and percentage of motile spermatozoa were not affected by BMI in their study. Similarly, in the present study, we did not observe significant differences among the groups in ejaculated volume and sperm morphology; however, sperm concentration was not different and a lower total motile count was observed in overweight and obese groups.

The impact of obesity on fertility can be attributed primarily to endocrine mechanisms [12,24]. It has been reported that higher BMI values are related to lower inhibin B levels [5,13] and that hyperthermia, resulting from accumulation of fatty tissue around the scrotum, causes oxidative stress to the testicles [15].

Reactive oxygen species (ROS) play an important role in modulating the essential functions of sperm, e.g. capacitation, hyperactivation and acrosomal reaction; however, imbalances between ROS production and semen antioxidant capacity will result in oxidation of membrane polyunsaturated fatty acids [25,26], loss of mitochondrial membrane potential [27], and single and double-strand DNA fragmentation [28].

There is a strong association between decreased sperm motility and oxidative stress [29,30]. Sperm motility is generated through constant ATP production by mitochondria localized in the sperm midpiece. To produce ATP through oxidative phosphorylation, the mitochondrial membrane must have selective permeability, which maintains an electrolytical gradient between the inner and outer mitochondrial environments. Excessive ROS will alter phospholipid membranes, and thus disrupt membrane selectivity, and will also inhibit oxidative phosphorylation, ultimately leading to decreased ATP production [31,32]. In the present study, men in the obese group had lower sperm mitochondrial activity than those in the eutrophic group.

Excessive production of ROS may also lead to increased DNA damage, through the production of lypid degradation by-products which bind to DNA, through oxidation of DNA bases (mainly guanosine), or through direct interaction with the DNA strand, leading to non-specific single- and double-strand breaks [28,29]. Sperm DNA integrity has been extensively researched recently as a marker of male factor infertility. Initially, ROS usually alter the sperm lipid bilayer by oxidation of methylene groups in polyunsaturated fatty acids. The process ultimately leads to the production of lipid degradation by-products, of which malonaldehyde is one of the most common. Malonaldehyde is an alkylating agent with the ability to covalently bind to nucleophilic groups of DNA, peptides and proteins, modifying their molecular function [32]. In the second stage of excessive ROS production, guanosine is oxidized to 8′-OH guanosine, a promutagenic effect that leads to altered pairing of DNA bases [28]. In the third stage, excess ROS will directly interact with DNA and cause single- and double-strand non-specific breaks in the DNA molecule [29].

In their study on 483 men, Chavarro et al.[33] observed that sperm with high DNA damage were significantly more numerous in obese men than in normal-weight men. In a study by Kort et al.[10] increasing BMI was positively correlated with the DNA fragmentation index calculated using the sperm chromatin structure assay. Obese and overweight men had a higher DNA fragmentation index (27% and 25.8%, respectively) than normal-weight men (19.9%). In the present study, we found that men in the obese group had a higher percentage of sperm with DNA damage than those in the eutrophic and overweight groups.

We conclude that higher BMI values are associated with decreased mitochondrial activity, progressive motility and increased DNA fragmentation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES