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Factors contributing to gender differences in serum retinol and α-tocopherol in infertile couples
RBMOnline - Vol 19. No 4. 2009 583–590 Reproductive BioMedicine Online; www.rbmonline.com/Article/4052 on web 19 August 2009
Article Factors contributing to gender differences in serum retinol and a-tocopherol in infertile couples Dr Majedah Al-Azemi FRCOG is Associate Professor at the Faculty of Medicine, Kuwait University. She undertook postgraduate training in United Kingdom and received a fellowship in Reproductive Medicine from Oxford University in 1999. She has published in the areas of infertility and recurrent pregnancy loss. In 2007, she spent time at the Academic Unit of Reproductive and Developmental Medicine, University of Sheffield on a sabbatical visit to research the molecular basis of ectopic pregnancy.
Dr Majedah Al-Azemi MK Al-Azemi1,2, AE Omu1, T Fatinikun1, N Mannazhath1, S Abraham1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Kuwait University, Kuwait 2 Correspondence: e-mail: [email protected]
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Abstract Oxidative stress is detrimental to fertility potential. Retinol and a-tocopherol are natural antioxidants that inhibit lipid peroxidation and protect against cell damage induced by oxidative stress. This study evaluated whether gender-related differences in antioxidant status exist among couples with infertility and, if so, to evaluate relevant factors that may contribute to such differences. Retinol and a-tocopherol in the sera of 40 couples and in the semen of 40 men were measured using high performance liquid chromatography. Serum retinol and a-tocopherol in women were significantly higher than in men (both P < 0.001). There was an inverse relationship between body mass index and serum retinol and a-tocopherol in both men and women. Heavy smokers (20 cigarettes/day), compared with nonsmokers, had lower serum concentrations of retinol and a-tocopherol. Serum concentrations of retinol and a-tocopherol in men with normal sperm parameters were significantly higher than in those with oligozoospermia and asthenozoospermia (both P < 0.001). Lower serum retinol and a-tocopherol in men compared with their female partners could be related to their older age, higher body mass index and smoking habits. Low concentrations of these natural antioxidants were associated with abnormal semen parameters in men and anovulation in women. Keywords: antioxidant status, gender differences, oxidative stress, retinal, a-tocopherol
Introduction Oxidative stress results from an imbalance between pro-oxidants (reactive oxygen species; ROS) and the body’s scavenging ability (antioxidants). Numerous studies have shown that oxidative stress plays a role in the pathophysiology of infertility in both male and female genders (Sikka, 2001). In females, ROS may have a regulatory role in oocyte maturation, folliculogenesis, ovarian steroidogenesis and luteolysis (Sabatini et al., 1999; Behrman et al., 2001). In males, ROS-induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility (Duru et al., 2000; Aitken and Krausz, 2001; Agarwal et al., 2004). Human spermatozoa have a high content of polysaturated fatty acids and a limited capacity for DNA repair, thus ren-
dering them extremely sensitive to attack by ROS (Menezo et al., 2007). ROS should be continuously inactivated to keep only a small amount necessary to maintain normal cell function (Pierce et al., 2004). Under normal conditions, scavenging molecules known as antioxidants convert ROS to H2O to prevent overproduction of ROS. There are two types of antioxidants in the human body: enzymatic antioxidants and non-enzymatic antioxidants (Allard et al., 1994). Enzymatic antioxidants include superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Non-enzymatic antioxidants include dietary supplements. The body’s complex antioxidant system is influenced by dietary intake of antioxidant vitamins and minerals such as vitamin C, vitamin
Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
E, selenium, zinc, glutathione, b-carotene, and carotene (Riley and Behrman, 1991). Vitamin A (retinol and b-carotene) and vitamin E (a-tocopherol) are natural lipid soluble antioxidants. Vegetables, fruits and vegetable oils, nuts, whole grain like wheat corn and broccoli are rich sources of both vitamins. They have a major role in the body defences against oxidative damage (Allard et al., 1994). Vitamin A includes retinol and its precursor b-carotene, which has one-sixth of the vitamin activity as equivalent retinol. Retinol and its synthetic derivatives are important for their potential utility as immunomodulatory agents and in the prevention of cancer and risk of coronary heart disease. b-Carotene is known to enhance both T- and B-cell mitogen responsiveness and promote the increase in the number of circulating T cells (CD3+) and in the helper T-cells (CD4+) subset in humans given a supplement of the vitamin (Alexander et al., 1985). Similarly, vitamin E has been shown to contribute to the body’s defence system. It is made up of eight related compounds, four of which are called tocopherol (alpha, beta, gamma and delta). The other four are called tocotrienol. D-a-Tocopherol is the natural vitamin E, and it is the most available in the body. It is better absorbed and retained in the body, and twice as much ends up in the bloodstream, than the synthetic version (di-a-tocopherol). As an antioxidant, a-tocopherol prevents chronic degenerative disease such as cancer, atherosclerosis and heart disease (Chow, 1991; Bostick et al., 1993; Knekt et al., 1994). Vitamin E is one of the body’s most important defences against free radical damage and boosting the immune system. Therefore, both vitamins A and E are crucial antioxidants that reduce disease risk by inhibiting the peroxidation of lipids and lipoproteins and contribute to the body defences against oxidative damage (Ames et al., 1993; Aksnes, 1994; Askari et al., 1994). Because antioxidant vitamins may affect an organism’s capacity for defence against reactive oxygen species, biological markers of the dietary exposure to these vitamins is of importance. The objective of this study was to evaluate whether genderrelated differences in antioxidant biological markers, namely retinol and a-tocopherol, exist among couples attending a combined infertility clinic and, if so, to evaluate the relevant factors that may contribute to such differences.
Materials and methods Patients
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This is cohort study involved 40 couples who attended the combined infertility clinic at the Maternity Hospital in Kuwait, with inability to achieve conception following more than 12 months of co-habitation. The study was approved by the Ethics Committee of the Faculty of Medicine and by the institutional review board of the Maternity Hospital. Written informed consent was obtained from all subjects. The study protocol included clinical evaluation, with history of the duration and type of infertility and smoking habits. Physical examination was carried out on both partners, including weight and height and body mass
index (BMI) was calculated (weight (kg)/height (m2)). Patients with hypertension, diabetes or any chronic condition were excluded from the study. The women had laparoscopy as a part of the infertility investigation for tubal patency and evaluation of ovulation.
Methodology Fasting blood samples, collected between 8:00 and 9:00 a.m., were centrifuged at 1000 rpm for 10 min, the serum was separated, stored, light protected, in a closed tube at 20°C until it was analysed for retinol and a-tocopherol and sex hormone profile.
Semen analysis All men had semen analysis after 3 days of sexual abstinence (World Health Organization, 1993). The semen analysis was carried out on fresh semen samples, and men were classified according to the sperm parameters: (i) normal sperm parameters, i.e. sperm count of 20 106/ml, with motility and normal morphology of 40%; (ii) oligozoospermia, i.e. sperm count of <20 106/ml; and (iii) asthenozoospermia, i.e. impaired motility with 40% being non-motile.
Estimation of sex hormones FSH, LH and testosterone were measured in both men and the women. In women, these hormones were measured on day 5 of their menstrual cycles. Serum progesterone was measured in women to evaluate ovulatory status. The timing of blood sampling was adjusted according to the patient’s cycle length. For women with regular 28-day cycles, blood samples were collected on day 21, otherwise serial measurements were taken to confirm anovulation. The hormones were estimated with Vidas parametric system strictly according to the guidelines in the manufacturer’s instruction booklet (bioMe´rieux, MO, USA) as previously described (Omu et al., 2003).
Chromatography The serum and seminal concentrations of retinol and a-tocopherol were estimated with high performance liquid chromatography (Waters Associates Inc, Milford Mass, USA); with pump, injector and UV detector of 292 nm filter (Millipore Corporation, Milford, MA, USA). The chromatography column was a reverse-phase C18 stainless steel column with dimensions 4 mm 11 cm. The standards included all-trans retinyl acetate and di-a-tocopherol (Sigma Chemical Co, St. Louis, MO, USA). The chromatography solvent was reagent grade methanol (95%) (Alltech Associates Inc., Deerfield, IL, USA). It was degassed before use. The standard curve was first developed with the standards and internal standards. In a typical chromatogram, the flow rate was 1.5 ml/min and total chromatographic run time of 15 min. Retinol and a-tocopherol peaked at 3.7 and 9.7 min, respectively. Thereafter the prepared serum was used. The injection volume used was 200 ll at 20°C and the duration of chromatographic analysis was 15 min. The time of separation of retinol and aRBMOnlineÒ
Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
tocopherol was automatically indicated on the chromatogram. The details of preparation of internal standards, serum and chromatography were carried out as previously described (Omu et al., 1999a).
to compare between more than two groups. Multiple linear regression was carried out using a-tocopherol and retinol as the dependent variables while gender, BMI and age were included as independent variables.
The concentration of retinol and a-tocopherol (X) was calculated as X = (Sa C)/St where Sa is the peak height of the vitamin in the sample, St is the height of the vitamin in the calibration standard and C is the concentration of the substance in the calibration standard. The intra-assay variation coefficient was 4.5% for retinol and 3.8% for atocopherol in serum. The detection limit and linearity range for retinol was 0.1–9.1 lmol/l and 0.9–29.5 lmol/l for a-tocopherol. Recovery was 96% for retinol and 98% for a-tocopherol as calculated from the internal standards.
Results
Measurements of other oxidative stress indices Malondialdehyde was measured using the thiobarbituric acid method as previously described (Kehinde et al., 2003). Superoxide dismutase, glutathione peroxidase and total antioxidant capacity were evaluated by enzyme-linked immunosorbent assay using commercially available kits as previously described (Omu et al., 1999b).
Statistical analysis Statistical analyses were carried out using Statistical Package for Social Sciences, version 15.0 (SPSS Inc, Chicago, USA). A probability level < 0.05 was used as the threshold for statistical significance. The quantitative variables are presented as means ± SD values and the qualitative variables as numbers and percentages. The chi-squared test was used to assess the association between two qualitative variables. Quantitative variables were compared between two independent groups using independent samples t-test, and one-way analysis of variance test (ANOVA) was used
Demographic characteristics and hormonal profile of the couples are shown in Table 1. The mean age of men was significantly higher than that of women (P < 0.001). The mean duration of infertility in men was significantly longer than that in women (P < 0.001) as some individuals had tried to conceive in previous marriages. More than 70% of both men and women had primary infertility. Similarly, more than 50% of both the men and women had BMI of 30 kg/m2 or more, showing that majority of patients were clinically obese. None of the women admitted to smoking cigarettes compared with 23 (57.5%) of the men who were smokers. Fourteen (35.0%) men were heavy smokers, smoking 20 or more cigarettes per day, 17 men (42.4%) had normal sperm parameters, 12 men (30.0%) had oligozoospermia and 11 (27.6%) had asthenozoospermia. Twenty-four (60.0%) women had regular menstrual cycles, with mid-luteal-phase serum progesterone of P10.0 nmol/l suggestive of ovulation. The remaining 16 (40.0%) women had anovulatory cycles. Twelve patients with anovulation fulfilled the Rotterdam criteria of polycystic ovary syndrome as previously described (Al-Azemi et al., 2004). The serum progesterone was significantly higher in the women with ovulatory cycles compared with those with anovulatory cycles (59.3 versus 2.5 nmol/l, P < 0.01). LH:FSH ratio (2.2 versus 0.8) and testosterone concentration (2.8 versus 1.1 nmol/l), were also significantly higher in the anovulatory women than their counterparts with ovulation (P < 0.05). The normal range of LH concentration is 2.2–12 IU/l in men and 2–15.4 IU/l in women, of FSH concentration is 0.9–9.8 IU/l in men and 2.5–9.0 IU/l
Table 1. Demographic characteristics and hormonal profile for 40 men and 40 women included in the study. Parameter
Men (n = 40)
Women (n = 40)
P value
Age (years) Infertility duration (years) Body mass index (kg/m2) LH (IU/l) FSH (IU/l) Testosterone (nmol/l) Type of infertility Primary Secondary Smoking Non-smoker <20 cigarettes/day 20 cigarettes/day
34.8 ± 8 7.2 ± 3.4 34.8 ± 4.2 5.4 ± 3.8 6.1 ± 4.5 14.9 ± 7.8
28.0 ± 4 4.4 ± 2.8 32 ± 3.6 7.6 ± 4.2 4.2 ± 3.1 1.8 ± 1.5
<0.001a <0.001a NSa <0.05a <0.05a <0.001a
31 (77.5) 9 (22.5)
29 (72.5) 11 (27.5)
NSb NSb
17 (42.5) 9 (22.5) 14 (35.0)
40 (100.0) 0 (0.0) 0 (0.0)
– – –
Values are means ± SD or n (%). NS, not statistically significant.
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a
Independent t-test.
b
Chi-squared test.
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Table 2. Oxidant/antioxidant status in 40 men and 40 women included in the study. Men (n = 40)
Women (n = 40)
P value
Antioxidants a-tocopherol (lmol/l) Retinol (lmol/l) Glutathione peroxidase (IU/ml) Total antioxidant capacity (mmol/l) Superoxide dismutase (IU/ml)
13.0 ± 2.5 4.3 ± 1.7 9.8 ± 2.4 0.88 ± 0.12 188 ± 14
19.0 ± 3.5a 7.6 ± 1.4a 10.2 ± 3.1 0.92 ± 0.04 168 ± 18a
<0.001 <0.001 NS NS <0.001
Oxidants Malondialdehyde (nmol/ml)
410 ± 180
284 ± 144a
<0.001
Values are means ± SD. NS, not statistically significant. a
Independent t-test.
in women, and of testosterone concentration is 10.5– 35.0 IU/l in men and 0.7–2.3 IU/l in women. The comparative serum concentrations of retinol and a-tocopherol in men and women are summarized in Table 2. The mean serum retinol concentration in women was significantly higher than that in men (7.6 ± 1.4 versus 4.3 ± 1.7 lmol/l, P < 0.001). The same pattern was observed for a-tocopherol (19 ± 3.5 versus 13 ± 2.5 lmol/ l, P < 0.001). Serum malondialdehyde was significantly higher among men compared with women (410 ± 180 versus 284 ± 144 nmol/ml, P < 0.001), while there were no significant differences in the enzymatic antioxidant glutathione peroxidase and total antioxidant capacity between men and women. Table 3 shows that, after controlling for variance accounted by BMI and age, the multiple linear regression analysis ascertained the significant relation of a-tocopherol and retinol with gender (overall significant regression, P < 0.001). The variables gender, BMI and age explained 59.8% of the variation in a-tocopherol and 54.6% of the variation in retinol (coefficient of determination, r2 = 0.598 and 0.546, respectively). Mean serum retinol concentration of 8.1 ± 0.6 lmol/l in the ovulatory women was significantly higher than 6.9 ± 0.8 lmol/l in the anovulatory women (P < 0.001).
Serum a-tocopherol revealed a similar pattern with 20.2 ± 4.4 lmol/l in ovulatory compared with 17.3 ± 3.8 lmol/l in anovulatory women (P = 0.03). Serum malondialdehyde was significantly higher among women with anovulatory cycles compared with those who experienced ovulation (P < 0.001). There were, however, no significant differences in total antioxidant capacity, glutathione peroxidase and superoxide dismutase in those with ovulatory compared with those with anovulatory cycles (Table 4). In Figure 1, the effect of cigarette smoking on serum concentrations of retinol and a-tocopherol was evaluated. There were significantly reduced retinol and a-tocopherol concentrations among smokers (all smokers) compared with non-smokers; 6.0 ± 1.5 lmol/l in non-smokers compared with 3.3 ± 1.2 lmol/l in smokers (P < 0.01) for retinol and 18.6 ± 2.5 versus 10.2 ± 2.8 lmol/l (P < 0.02) for a-tocopherol. This was manifested in dose-dependent pattern. Heavy smoking (20 cigarettes/day), was associated with significantly lower serum retinol and a-tocopherol concentrations than mild/moderate smokers (<20 cigarettes/ day) 2.8 ± 1.4 versus 4.6 ± 1.9 lmol/l for retinol (P < 0.001) and 8.2 ± 2.3 versus 12.8 ± 1.7 lmol/l for atocopherol (P < 0.001). As summarized in Figure 2, retinol and a-tocopherol concentrations were higher in women than men in all
Table 3. Standardized coefficients of independent variables from multiple linear regression analysis using a-tocopherol and retinol as the dependent variables.
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Variable
Standardized coefficient (b)
P value
Coefficient of determination (r2)
a-tocopherol Gender Body mass index Age
0.703 0.161 0.005
<0.001 0.043 NS
0.598
Retinol Gender Body mass index Age
0.762 0.106 0.010
<0.001 NS NS
0.546
NS, not statistically significant.
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Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
Table 4. Oxidant/antioxidant status in 40 women according to ovulatory status.
a-tocopherol (lmol/l) Retinol (lmol/l) Glutathione peroxidase (IU/ml) Total antioxidant capacity (mmol/l) Superoxide dismutase (IU/ml) Malondialdehyde (nmol/ml)
Ovulation (n = 24)
Anovulation (n = 16)
P valuea
20.2 ± 4.4 8.1 ± 0.6 10.4 ± 2.8 1.2± 0.8 192 ± 20 252 ± 120
17.3 ± 3.8* 6.9 ± 0.8*** 9.8 ± 2.2 0.82 ± 0.6 182 ± 14 480 ± 120***
<0.05 <0.001 NS NS NS <0.001
Values are means ± SD. a
Independent t-test.
Figure 1. The effect of smoking status on retinol and atocopherol concentrations in men. Results are expressed as means ± SD. Statistical analysis was by one-way analysis of variance.
BMI subgroups. a-tocopherol concentrations were significantly higher in the women than men for all BMI categories (P < 0.001). Retinol concentrations were significantly higher in women than men at BMI < 25 kg/m2 (P < 0.05) and BMI P 30 kg/m2 (P < 0.01). In both genders, increasing BMI had an inverse relationship with retinol and
a-tocopherol concentrations (P < 0.001). As BMI increased, in women, retinol concentration decreased from 8.2 to 4.4 lmol/l while a-tocopherol decreased from 28.4 to 16.8 lmol/l, while in men, retinol concentration decreased from 5.6 to 3.0 lmol/l and a-tocopherol decreased from 19.2 to 8.2 lmol/l.
Figure 2. The effect of body mass index on serum retinol and a-tocopherol in both genders. Results are expressed as means ± SD. P < 0.001 (one-way analysis of variance) for both variables among different BMI categories in both genders. P < 0.001 for a-tocopherol concentrations in women versus men in all BMI categories. Retinol concentrations were significantly higher in women than men at BMI < 25 kg/m2 (P < 0.05) and BMI P 30 kg/m2 (P < 0.01). RBMOnlineÒ
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Figure 3. Serum and seminal concentrations of retinol and a-tocopherol according to semen parameters. Results are expressed as means ± SD. Statistical analysis was by one-way analysis of variance.
The relationship between the serum concentrations of retinol and a-tocopherol and the sperm parameters in the men was evaluated (Figure 3). The serum concentrations of both retinol and a-tocopherol were significantly higher in men with normal sperm parameters compared with those with sperm dysfunction: retinol: normozoospermia (5.2 ± 1.2 lmol/l) compared with oligozoospermia (2.8 ± 0.6 lmol/l, P < 0.05), and asthenozoospermia (2.6 ± 0.4 lmol/l, P < 0.02); a-tocopherol: normozoospermia (18.8 ± 1.1 lmol/l) compared with oligozoospermia (10.2 ± 0.7 lmol/l, P < 0.05), and asthenozoospermia (8.8 ± 0.8 lmol/l, P < 0.01). Analysis of the seminal concentrations of retinol and a-tocopherol showed conflicting results. There was no significant difference in seminal retinol concentrations between men with normozoospermia and sperm dysfunction, while seminal a-tocopherol was significantly higher in men with normozoospermia (4.2 lmol/l) compared with oligozoospermia (2.6 lmol/l, P < 0.05) and asthenozoospermia (2.8 lmol/l, P < 0.05).
Discussion
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This study showed that gender-related differences in serum retinol and a-tocopherol exist, being significantly higher in women than in men. Among the factors that may contribute to gender difference are age, obesity and smoking. This is in agreement with other reports (Bolton-Smith et al., 1991; Ahmed et al., 1992), which have shown that factors such as age, sex, BMI, socio-economic status, medication, and vitamin supplementation are known to influence plasma antioxidant nutrient concentrations. Cigarette smoking is an important factor that can tip the balance in the direction of oxidative stress. Cigarette smoking is a good model of chronic oxidative stress. Tobacco smoke contains a variety of oxidizing agents (Pryor, 1982), which induce lipid peroxidation. This results in oxidative cellular damage (Halliwell, 1987; Rahman and MacNee, 1996) and is believed to contribute to the pathogenesis of germ cell apoptosis. In agreement with these findings is the mounting evidence that the concentrations of natural antioxidants retinol and a-tocopherol in the blood stream are considerably reduced by cigarette smoking (Bridges et al.,
1990; Faruque et al., 1995). Researchers from the National Institute of Health and Environmental Protection of the Netherlands assessed the relationship between smoking habits and dietary intake of antioxidant nutrients and their rich food sources. Men who smoked more than 20 cigarettes, had lower serum concentration of retinol and vitamin C than those who never smoked, in spite of high intake of food sources rich in antioxidants. This relationship was most dramatic for the male heavy smokers (Zondervan et al., 1996), as shown in the present study, demonstrating a dose-dependent effect. This effect can be explained by increased utilization of antioxidant vitamins during neutralization of phagocyte-derived free radicals (Maderazo et al., 1990). Several studies showed that supraphysiological concentrations of vitamin C, a-tocopherol and b-carotene can reduce lipid peroxidation induced by cigarette smoking (Allard et al., 1994). This was confirmed at the cellular level, where these vitamins neutralized extracellular hypochlorous acid generated by activated neutrophils in vitro (Anderson et al., 1990). Cigarette smoking is a common habit for men attending an infertility clinic in our community with an adverse effect on sperm parameters (Omu et al., 1998). The association between low serum concentrations of retinol and a-tocopherol and low seminal concentrations of a-tocopherol and sperm dysfunction has important clinical implications in infertility. A previous study demonstrated low total antioxidant activity, retinol and a-tocopherol in men with seminal infections (Omu et al., 1998). In men with prostatitis and vesiculitis, defective spermatozoa and leukocytes generate large amounts of ROS (Aitken et al., 1992; Kessopoulou et al., 1992; Rajasekaran et al., 1995). Reduced forms of oxygen, such as superoxide radicals, hydroxyl radicals and hydrogen peroxide, are generated during aerobic metabolism. To counter the effects of ROS, cells possess antioxidants as scavengers of the free radicals. The peroxidative chain reaction of polyunsaturated lipids can be terminated by retinol and a-tocopherol, water-soluble antioxidants. Vitamin C recycles oxidized a-tocopherol (Chan, 1993; Coudary et al., 1995). With vitamin C, vitamin E has been shown to enhance cytokine production by the regeneration of its active form after it has RBMOnlineÒ
Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
reacted with a free radical (Riley and Behrman, 1991). Menezo et al. (2007) evaluated the impact of antioxidant treatment on sperm DNA fragmentation and sperm head decondensation. Antioxidant treatment led to a decrease in sperm DNA fragmentation. However, it also led to an unexpected negative effect by increasing sperm decondensation. In an extensive review by Agarwal et al. (2004), the efficacy of antioxidants in male infertility was evaluated. Many clinical trials have shown the positive effects of antioxidants on male infertility; however, these studies have used different forms in different combinations and dosages for varying periods of time. Therefore, the evidence supporting antioxidant use does exist but a consensus is required regarding type and dosage of antioxidants to be used. Another interesting finding in the present study was the significantly reduced serum retinol and a-tocopherol concentrations in anovulatory women compared with those with ovulatory cycles. Most women having low serum retinol and a-tocopherol in the anovulatory group have polycystic ovary syndrome, with high circulating androgen, testosterone and LH:FSH ratio greater than 2. In a study conducted on postmenopausal women before and after hormone replacement therapy and 20 premenopausal women who served as controls, data showed that oestrogens do not reduce plasma a-tocopherol concentrations (Wu et al., 1996). The present study has demonstrated indirect evidence that women with high circulating androgen have lower serum retinol and a-tocopherol concentrations. Obesity is another factor associated with increased oxidative stress. Few studies have investigated the antioxidant system in obese patients with infertility. The findings in this study have demonstrated the effect of BMI on antioxidant status. Previous studies showed that obesity elevates oxidative stress by elevations in lipid peroxidation (malondialdehyde, hydroperoxides, 4-hydroxynonenal, isoprostanes, and conjugated dienes) or protein oxidation (8-hydroxydeoxyguanosine). Lipid peroxidation is associated with a low systemic antioxidant defence (i.e. antioxidant enzymes, tissue dietary antioxidants) as confirmed in this study. These findings are of clinical relevance in an infertility clinic setting. Obesity-related oxidative stress may be corrected by improving antioxidant defences through fat volume reduction via surgery, pharmacological agents, exercise and/or dietary modification such as vitamin A and E supplementation (Vincent et al., 2007). There are many dire long-term clinical implications of these findings from the perspective of infertility management. Men with sperm dysfunction and women with anovulation, especially if they are smokers and obese, could benefit from simple measures such as weight reduction programmes, smoking cessation and antioxidant supplements. The practice of actively treating these patients, who have an extreme desire for childbearing, without starting with such simple measures, may need revision. More research is certainly needed to further explore the association between the serum concentrations of retinol and a-tocopherol and infertilityrelated problems. Furthermore, there is a need to establish RBMOnlineÒ
the required preventive and therapeutic doses of these antioxidant nutrients in the management of such problems.
Acknowledgements The authors sincerely appreciate the contribution of Dr. Clifford Abiaka of Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences and Nursing, Kuwait University, in donating the standards used in the project.
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Chan AC 1993 Partners in defense, vitamin E and vitamin C. Canadian Journal of Physiology and Pharmacology 71, 725–731. Chow CK 1991 Vitamin E and oxidative stress. Free Radical Biology and Medicine 11, 215–232. Coudary C, Hida H, Boucher F et al. 1995 Modulation by selenium supplementation of lipid peroxidation induced by chronic administration of adriamycin in rats. Nutrition 11, 512–516. Duru NK, Morshedi M, Oehninger S 2000 Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertility and Sterility 74, 1200–1207. Faruque O, Khan MR, Rahman M et al. 1995 Relationship between smoking and antioxidant status. British Journal of Nutrition 73, 625–632. Halliwell B 1987 Oxidants and human disease: some new concepts. The Federation of American Societies for Experimental Biology Journal 1, 358–364. Kehinde EO, Mojiminiyi OA, Mahmoud AH et al. 2003 The significance of measuring the time course of serum malondialdehyde concentration in patients with torsion of the testis. Journal of Urology 169, 2177–2180. Kessopoulou E, Tomlinson MJ, Barrat CLR et al. 1992 Origin of reactive oxygen species in human semen spermatozoa or leukocytes. Journal of Reproduction and Fertility 94, 463–470. Knekt P, Reunanem A, Jarvinen R et al. 1994 Antioxidant vitamin intake and coronary mortality in a longitudinal population study. American Journal of Epidemiology 139, 1180–1189. Maderazo EG, Woronick CL, Hickingbotham N et al. 1990 Additional evidence of autoxidation as a possible mechanism of neutrophil locomotory dysfunction in blunt trauma. Critical Care Medicine 18, 141–147. Menezo Y, Hazout A, Panteix G et al. 2007 Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reproductive BioMedicine Online 14, 418–421. Omu AE, Al-Azemi MK, Makhseed M et al. 2003 Differential expression of T-helper cytokines in the peritoneal fluid of women with normal ovarian cycle compared with women with chronic anovulation. Acta Obstetricia et Gynecologica Scandinavica 82, 603–609. Omu AE, Fatinikun T, Mannazhath N et al. 1999 Significance of simultaneous determination of serum and seminal plasma alpha-tocopherol and retinol in infertile men by high performance liquid chromatography. Andrologia 31, 347–354. Omu AE, Al-Qattan F, Abdelhadi F et al. 1999 Seminal immune response in infertile men with leukocytospermia: effect on antioxidant activity. European Journal of Obstetrics and Gynecology and Reproductive Biology 79, 179–184.
Omu AE, Al-Othman S, Mohammad AS et al. 1998 Antibiotic therapy of seminal infection: effect on antioxidant activity and T helper cytokines. Journal of Reproductive Medicine 43, 857–864. Pierce JD, Cackler AB, Arnett MG 2004 Why should you care about free radicals? RN: National Magazine for Nursing 67, 38–42. Pryor WA 1982 Free radical biology: xenobiotics, cancer, and aging. Annals of the New York Academy of Sciences 393, 1–22. Rahman I, MacNee W 1996 Role of oxidants in smoking induced lung diseases. Free Radical Biology and Medicine 21, 669–681. Rajasekaran M, Hallstrom WJG, Naz RK et al. 1995 Oxidative stress and interleukins in seminal plasma during leukocytospermia. Fertility and Sterility 64, 166–171. Riley JCM, Behrman HR 1991 Oxygen radicals and reactive oxygen species in reproduction. Proceedings of the Society for Experimental Biology and Medicine 198, 781–791. Sabatini L, Wilson C, Lower A et al. 1999 Superoxide dismutase activity in human follicular fluid after controlled ovarian hyperstimulation in women undergoing in vitro fertilization. Fertility and Sterility 72, 1027–1034. Sikka SC 2001 Relative impact of oxidative stress on male reproductive function. Current Medicinal Chemistry 8, 851–862. Vincent HK, Innes KE, Vincent KR 2007 Oxidative stress and potential interventions to reduce oxidative stress in overweight and obesity. Diabetes, Obesity and Metabolism 9, 813–839. World Health Organization 1993. WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction 3rd edn. Cambridge University Press, New York, 5. Wu J, Norris LA, Wen YC et al. 1996 The effects of hormone replacement therapy on plasma vitamin E levels in postmenopausal women. European Journal of Obstetrics and Gynecology and Reproductive Biology 66, 151–154. Zondervan KT, Ocke MC, Smit HA et al. 1996 Do dietary and supplementary intakes of antioxidants differ with smoking status? International Journal of Epidemiology 25, 70–79.
Declaration: The authors report no financial or commercial conflicts of interest. Received 16 November 2008; refereed 17 December 2008; accepted 11 May 2009.
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Article Factors contributing to gender differences in serum retinol and a-tocopherol in infertile couples Dr Majedah Al-Azemi FRCOG is Associate Professor at the Faculty of Medicine, Kuwait University. She undertook postgraduate training in United Kingdom and received a fellowship in Reproductive Medicine from Oxford University in 1999. She has published in the areas of infertility and recurrent pregnancy loss. In 2007, she spent time at the Academic Unit of Reproductive and Developmental Medicine, University of Sheffield on a sabbatical visit to research the molecular basis of ectopic pregnancy.
Dr Majedah Al-Azemi MK Al-Azemi1,2, AE Omu1, T Fatinikun1, N Mannazhath1, S Abraham1 Department of Obstetrics and Gynaecology, Faculty of Medicine, Kuwait University, Kuwait 2 Correspondence: e-mail: [email protected]
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Abstract Oxidative stress is detrimental to fertility potential. Retinol and a-tocopherol are natural antioxidants that inhibit lipid peroxidation and protect against cell damage induced by oxidative stress. This study evaluated whether gender-related differences in antioxidant status exist among couples with infertility and, if so, to evaluate relevant factors that may contribute to such differences. Retinol and a-tocopherol in the sera of 40 couples and in the semen of 40 men were measured using high performance liquid chromatography. Serum retinol and a-tocopherol in women were significantly higher than in men (both P < 0.001). There was an inverse relationship between body mass index and serum retinol and a-tocopherol in both men and women. Heavy smokers (20 cigarettes/day), compared with nonsmokers, had lower serum concentrations of retinol and a-tocopherol. Serum concentrations of retinol and a-tocopherol in men with normal sperm parameters were significantly higher than in those with oligozoospermia and asthenozoospermia (both P < 0.001). Lower serum retinol and a-tocopherol in men compared with their female partners could be related to their older age, higher body mass index and smoking habits. Low concentrations of these natural antioxidants were associated with abnormal semen parameters in men and anovulation in women. Keywords: antioxidant status, gender differences, oxidative stress, retinal, a-tocopherol
Introduction Oxidative stress results from an imbalance between pro-oxidants (reactive oxygen species; ROS) and the body’s scavenging ability (antioxidants). Numerous studies have shown that oxidative stress plays a role in the pathophysiology of infertility in both male and female genders (Sikka, 2001). In females, ROS may have a regulatory role in oocyte maturation, folliculogenesis, ovarian steroidogenesis and luteolysis (Sabatini et al., 1999; Behrman et al., 2001). In males, ROS-induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility (Duru et al., 2000; Aitken and Krausz, 2001; Agarwal et al., 2004). Human spermatozoa have a high content of polysaturated fatty acids and a limited capacity for DNA repair, thus ren-
dering them extremely sensitive to attack by ROS (Menezo et al., 2007). ROS should be continuously inactivated to keep only a small amount necessary to maintain normal cell function (Pierce et al., 2004). Under normal conditions, scavenging molecules known as antioxidants convert ROS to H2O to prevent overproduction of ROS. There are two types of antioxidants in the human body: enzymatic antioxidants and non-enzymatic antioxidants (Allard et al., 1994). Enzymatic antioxidants include superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Non-enzymatic antioxidants include dietary supplements. The body’s complex antioxidant system is influenced by dietary intake of antioxidant vitamins and minerals such as vitamin C, vitamin
Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
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E, selenium, zinc, glutathione, b-carotene, and carotene (Riley and Behrman, 1991). Vitamin A (retinol and b-carotene) and vitamin E (a-tocopherol) are natural lipid soluble antioxidants. Vegetables, fruits and vegetable oils, nuts, whole grain like wheat corn and broccoli are rich sources of both vitamins. They have a major role in the body defences against oxidative damage (Allard et al., 1994). Vitamin A includes retinol and its precursor b-carotene, which has one-sixth of the vitamin activity as equivalent retinol. Retinol and its synthetic derivatives are important for their potential utility as immunomodulatory agents and in the prevention of cancer and risk of coronary heart disease. b-Carotene is known to enhance both T- and B-cell mitogen responsiveness and promote the increase in the number of circulating T cells (CD3+) and in the helper T-cells (CD4+) subset in humans given a supplement of the vitamin (Alexander et al., 1985). Similarly, vitamin E has been shown to contribute to the body’s defence system. It is made up of eight related compounds, four of which are called tocopherol (alpha, beta, gamma and delta). The other four are called tocotrienol. D-a-Tocopherol is the natural vitamin E, and it is the most available in the body. It is better absorbed and retained in the body, and twice as much ends up in the bloodstream, than the synthetic version (di-a-tocopherol). As an antioxidant, a-tocopherol prevents chronic degenerative disease such as cancer, atherosclerosis and heart disease (Chow, 1991; Bostick et al., 1993; Knekt et al., 1994). Vitamin E is one of the body’s most important defences against free radical damage and boosting the immune system. Therefore, both vitamins A and E are crucial antioxidants that reduce disease risk by inhibiting the peroxidation of lipids and lipoproteins and contribute to the body defences against oxidative damage (Ames et al., 1993; Aksnes, 1994; Askari et al., 1994). Because antioxidant vitamins may affect an organism’s capacity for defence against reactive oxygen species, biological markers of the dietary exposure to these vitamins is of importance. The objective of this study was to evaluate whether genderrelated differences in antioxidant biological markers, namely retinol and a-tocopherol, exist among couples attending a combined infertility clinic and, if so, to evaluate the relevant factors that may contribute to such differences.
Materials and methods Patients
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This is cohort study involved 40 couples who attended the combined infertility clinic at the Maternity Hospital in Kuwait, with inability to achieve conception following more than 12 months of co-habitation. The study was approved by the Ethics Committee of the Faculty of Medicine and by the institutional review board of the Maternity Hospital. Written informed consent was obtained from all subjects. The study protocol included clinical evaluation, with history of the duration and type of infertility and smoking habits. Physical examination was carried out on both partners, including weight and height and body mass
index (BMI) was calculated (weight (kg)/height (m2)). Patients with hypertension, diabetes or any chronic condition were excluded from the study. The women had laparoscopy as a part of the infertility investigation for tubal patency and evaluation of ovulation.
Methodology Fasting blood samples, collected between 8:00 and 9:00 a.m., were centrifuged at 1000 rpm for 10 min, the serum was separated, stored, light protected, in a closed tube at 20°C until it was analysed for retinol and a-tocopherol and sex hormone profile.
Semen analysis All men had semen analysis after 3 days of sexual abstinence (World Health Organization, 1993). The semen analysis was carried out on fresh semen samples, and men were classified according to the sperm parameters: (i) normal sperm parameters, i.e. sperm count of 20 106/ml, with motility and normal morphology of 40%; (ii) oligozoospermia, i.e. sperm count of <20 106/ml; and (iii) asthenozoospermia, i.e. impaired motility with 40% being non-motile.
Estimation of sex hormones FSH, LH and testosterone were measured in both men and the women. In women, these hormones were measured on day 5 of their menstrual cycles. Serum progesterone was measured in women to evaluate ovulatory status. The timing of blood sampling was adjusted according to the patient’s cycle length. For women with regular 28-day cycles, blood samples were collected on day 21, otherwise serial measurements were taken to confirm anovulation. The hormones were estimated with Vidas parametric system strictly according to the guidelines in the manufacturer’s instruction booklet (bioMe´rieux, MO, USA) as previously described (Omu et al., 2003).
Chromatography The serum and seminal concentrations of retinol and a-tocopherol were estimated with high performance liquid chromatography (Waters Associates Inc, Milford Mass, USA); with pump, injector and UV detector of 292 nm filter (Millipore Corporation, Milford, MA, USA). The chromatography column was a reverse-phase C18 stainless steel column with dimensions 4 mm 11 cm. The standards included all-trans retinyl acetate and di-a-tocopherol (Sigma Chemical Co, St. Louis, MO, USA). The chromatography solvent was reagent grade methanol (95%) (Alltech Associates Inc., Deerfield, IL, USA). It was degassed before use. The standard curve was first developed with the standards and internal standards. In a typical chromatogram, the flow rate was 1.5 ml/min and total chromatographic run time of 15 min. Retinol and a-tocopherol peaked at 3.7 and 9.7 min, respectively. Thereafter the prepared serum was used. The injection volume used was 200 ll at 20°C and the duration of chromatographic analysis was 15 min. The time of separation of retinol and aRBMOnlineÒ
Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
tocopherol was automatically indicated on the chromatogram. The details of preparation of internal standards, serum and chromatography were carried out as previously described (Omu et al., 1999a).
to compare between more than two groups. Multiple linear regression was carried out using a-tocopherol and retinol as the dependent variables while gender, BMI and age were included as independent variables.
The concentration of retinol and a-tocopherol (X) was calculated as X = (Sa C)/St where Sa is the peak height of the vitamin in the sample, St is the height of the vitamin in the calibration standard and C is the concentration of the substance in the calibration standard. The intra-assay variation coefficient was 4.5% for retinol and 3.8% for atocopherol in serum. The detection limit and linearity range for retinol was 0.1–9.1 lmol/l and 0.9–29.5 lmol/l for a-tocopherol. Recovery was 96% for retinol and 98% for a-tocopherol as calculated from the internal standards.
Results
Measurements of other oxidative stress indices Malondialdehyde was measured using the thiobarbituric acid method as previously described (Kehinde et al., 2003). Superoxide dismutase, glutathione peroxidase and total antioxidant capacity were evaluated by enzyme-linked immunosorbent assay using commercially available kits as previously described (Omu et al., 1999b).
Statistical analysis Statistical analyses were carried out using Statistical Package for Social Sciences, version 15.0 (SPSS Inc, Chicago, USA). A probability level < 0.05 was used as the threshold for statistical significance. The quantitative variables are presented as means ± SD values and the qualitative variables as numbers and percentages. The chi-squared test was used to assess the association between two qualitative variables. Quantitative variables were compared between two independent groups using independent samples t-test, and one-way analysis of variance test (ANOVA) was used
Demographic characteristics and hormonal profile of the couples are shown in Table 1. The mean age of men was significantly higher than that of women (P < 0.001). The mean duration of infertility in men was significantly longer than that in women (P < 0.001) as some individuals had tried to conceive in previous marriages. More than 70% of both men and women had primary infertility. Similarly, more than 50% of both the men and women had BMI of 30 kg/m2 or more, showing that majority of patients were clinically obese. None of the women admitted to smoking cigarettes compared with 23 (57.5%) of the men who were smokers. Fourteen (35.0%) men were heavy smokers, smoking 20 or more cigarettes per day, 17 men (42.4%) had normal sperm parameters, 12 men (30.0%) had oligozoospermia and 11 (27.6%) had asthenozoospermia. Twenty-four (60.0%) women had regular menstrual cycles, with mid-luteal-phase serum progesterone of P10.0 nmol/l suggestive of ovulation. The remaining 16 (40.0%) women had anovulatory cycles. Twelve patients with anovulation fulfilled the Rotterdam criteria of polycystic ovary syndrome as previously described (Al-Azemi et al., 2004). The serum progesterone was significantly higher in the women with ovulatory cycles compared with those with anovulatory cycles (59.3 versus 2.5 nmol/l, P < 0.01). LH:FSH ratio (2.2 versus 0.8) and testosterone concentration (2.8 versus 1.1 nmol/l), were also significantly higher in the anovulatory women than their counterparts with ovulation (P < 0.05). The normal range of LH concentration is 2.2–12 IU/l in men and 2–15.4 IU/l in women, of FSH concentration is 0.9–9.8 IU/l in men and 2.5–9.0 IU/l
Table 1. Demographic characteristics and hormonal profile for 40 men and 40 women included in the study. Parameter
Men (n = 40)
Women (n = 40)
P value
Age (years) Infertility duration (years) Body mass index (kg/m2) LH (IU/l) FSH (IU/l) Testosterone (nmol/l) Type of infertility Primary Secondary Smoking Non-smoker <20 cigarettes/day 20 cigarettes/day
34.8 ± 8 7.2 ± 3.4 34.8 ± 4.2 5.4 ± 3.8 6.1 ± 4.5 14.9 ± 7.8
28.0 ± 4 4.4 ± 2.8 32 ± 3.6 7.6 ± 4.2 4.2 ± 3.1 1.8 ± 1.5
<0.001a <0.001a NSa <0.05a <0.05a <0.001a
31 (77.5) 9 (22.5)
29 (72.5) 11 (27.5)
NSb NSb
17 (42.5) 9 (22.5) 14 (35.0)
40 (100.0) 0 (0.0) 0 (0.0)
– – –
Values are means ± SD or n (%). NS, not statistically significant.
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a
Independent t-test.
b
Chi-squared test.
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Table 2. Oxidant/antioxidant status in 40 men and 40 women included in the study. Men (n = 40)
Women (n = 40)
P value
Antioxidants a-tocopherol (lmol/l) Retinol (lmol/l) Glutathione peroxidase (IU/ml) Total antioxidant capacity (mmol/l) Superoxide dismutase (IU/ml)
13.0 ± 2.5 4.3 ± 1.7 9.8 ± 2.4 0.88 ± 0.12 188 ± 14
19.0 ± 3.5a 7.6 ± 1.4a 10.2 ± 3.1 0.92 ± 0.04 168 ± 18a
<0.001 <0.001 NS NS <0.001
Oxidants Malondialdehyde (nmol/ml)
410 ± 180
284 ± 144a
<0.001
Values are means ± SD. NS, not statistically significant. a
Independent t-test.
in women, and of testosterone concentration is 10.5– 35.0 IU/l in men and 0.7–2.3 IU/l in women. The comparative serum concentrations of retinol and a-tocopherol in men and women are summarized in Table 2. The mean serum retinol concentration in women was significantly higher than that in men (7.6 ± 1.4 versus 4.3 ± 1.7 lmol/l, P < 0.001). The same pattern was observed for a-tocopherol (19 ± 3.5 versus 13 ± 2.5 lmol/ l, P < 0.001). Serum malondialdehyde was significantly higher among men compared with women (410 ± 180 versus 284 ± 144 nmol/ml, P < 0.001), while there were no significant differences in the enzymatic antioxidant glutathione peroxidase and total antioxidant capacity between men and women. Table 3 shows that, after controlling for variance accounted by BMI and age, the multiple linear regression analysis ascertained the significant relation of a-tocopherol and retinol with gender (overall significant regression, P < 0.001). The variables gender, BMI and age explained 59.8% of the variation in a-tocopherol and 54.6% of the variation in retinol (coefficient of determination, r2 = 0.598 and 0.546, respectively). Mean serum retinol concentration of 8.1 ± 0.6 lmol/l in the ovulatory women was significantly higher than 6.9 ± 0.8 lmol/l in the anovulatory women (P < 0.001).
Serum a-tocopherol revealed a similar pattern with 20.2 ± 4.4 lmol/l in ovulatory compared with 17.3 ± 3.8 lmol/l in anovulatory women (P = 0.03). Serum malondialdehyde was significantly higher among women with anovulatory cycles compared with those who experienced ovulation (P < 0.001). There were, however, no significant differences in total antioxidant capacity, glutathione peroxidase and superoxide dismutase in those with ovulatory compared with those with anovulatory cycles (Table 4). In Figure 1, the effect of cigarette smoking on serum concentrations of retinol and a-tocopherol was evaluated. There were significantly reduced retinol and a-tocopherol concentrations among smokers (all smokers) compared with non-smokers; 6.0 ± 1.5 lmol/l in non-smokers compared with 3.3 ± 1.2 lmol/l in smokers (P < 0.01) for retinol and 18.6 ± 2.5 versus 10.2 ± 2.8 lmol/l (P < 0.02) for a-tocopherol. This was manifested in dose-dependent pattern. Heavy smoking (20 cigarettes/day), was associated with significantly lower serum retinol and a-tocopherol concentrations than mild/moderate smokers (<20 cigarettes/ day) 2.8 ± 1.4 versus 4.6 ± 1.9 lmol/l for retinol (P < 0.001) and 8.2 ± 2.3 versus 12.8 ± 1.7 lmol/l for atocopherol (P < 0.001). As summarized in Figure 2, retinol and a-tocopherol concentrations were higher in women than men in all
Table 3. Standardized coefficients of independent variables from multiple linear regression analysis using a-tocopherol and retinol as the dependent variables.
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Variable
Standardized coefficient (b)
P value
Coefficient of determination (r2)
a-tocopherol Gender Body mass index Age
0.703 0.161 0.005
<0.001 0.043 NS
0.598
Retinol Gender Body mass index Age
0.762 0.106 0.010
<0.001 NS NS
0.546
NS, not statistically significant.
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Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
Table 4. Oxidant/antioxidant status in 40 women according to ovulatory status.
a-tocopherol (lmol/l) Retinol (lmol/l) Glutathione peroxidase (IU/ml) Total antioxidant capacity (mmol/l) Superoxide dismutase (IU/ml) Malondialdehyde (nmol/ml)
Ovulation (n = 24)
Anovulation (n = 16)
P valuea
20.2 ± 4.4 8.1 ± 0.6 10.4 ± 2.8 1.2± 0.8 192 ± 20 252 ± 120
17.3 ± 3.8* 6.9 ± 0.8*** 9.8 ± 2.2 0.82 ± 0.6 182 ± 14 480 ± 120***
<0.05 <0.001 NS NS NS <0.001
Values are means ± SD. a
Independent t-test.
Figure 1. The effect of smoking status on retinol and atocopherol concentrations in men. Results are expressed as means ± SD. Statistical analysis was by one-way analysis of variance.
BMI subgroups. a-tocopherol concentrations were significantly higher in the women than men for all BMI categories (P < 0.001). Retinol concentrations were significantly higher in women than men at BMI < 25 kg/m2 (P < 0.05) and BMI P 30 kg/m2 (P < 0.01). In both genders, increasing BMI had an inverse relationship with retinol and
a-tocopherol concentrations (P < 0.001). As BMI increased, in women, retinol concentration decreased from 8.2 to 4.4 lmol/l while a-tocopherol decreased from 28.4 to 16.8 lmol/l, while in men, retinol concentration decreased from 5.6 to 3.0 lmol/l and a-tocopherol decreased from 19.2 to 8.2 lmol/l.
Figure 2. The effect of body mass index on serum retinol and a-tocopherol in both genders. Results are expressed as means ± SD. P < 0.001 (one-way analysis of variance) for both variables among different BMI categories in both genders. P < 0.001 for a-tocopherol concentrations in women versus men in all BMI categories. Retinol concentrations were significantly higher in women than men at BMI < 25 kg/m2 (P < 0.05) and BMI P 30 kg/m2 (P < 0.01). RBMOnlineÒ
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Figure 3. Serum and seminal concentrations of retinol and a-tocopherol according to semen parameters. Results are expressed as means ± SD. Statistical analysis was by one-way analysis of variance.
The relationship between the serum concentrations of retinol and a-tocopherol and the sperm parameters in the men was evaluated (Figure 3). The serum concentrations of both retinol and a-tocopherol were significantly higher in men with normal sperm parameters compared with those with sperm dysfunction: retinol: normozoospermia (5.2 ± 1.2 lmol/l) compared with oligozoospermia (2.8 ± 0.6 lmol/l, P < 0.05), and asthenozoospermia (2.6 ± 0.4 lmol/l, P < 0.02); a-tocopherol: normozoospermia (18.8 ± 1.1 lmol/l) compared with oligozoospermia (10.2 ± 0.7 lmol/l, P < 0.05), and asthenozoospermia (8.8 ± 0.8 lmol/l, P < 0.01). Analysis of the seminal concentrations of retinol and a-tocopherol showed conflicting results. There was no significant difference in seminal retinol concentrations between men with normozoospermia and sperm dysfunction, while seminal a-tocopherol was significantly higher in men with normozoospermia (4.2 lmol/l) compared with oligozoospermia (2.6 lmol/l, P < 0.05) and asthenozoospermia (2.8 lmol/l, P < 0.05).
Discussion
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This study showed that gender-related differences in serum retinol and a-tocopherol exist, being significantly higher in women than in men. Among the factors that may contribute to gender difference are age, obesity and smoking. This is in agreement with other reports (Bolton-Smith et al., 1991; Ahmed et al., 1992), which have shown that factors such as age, sex, BMI, socio-economic status, medication, and vitamin supplementation are known to influence plasma antioxidant nutrient concentrations. Cigarette smoking is an important factor that can tip the balance in the direction of oxidative stress. Cigarette smoking is a good model of chronic oxidative stress. Tobacco smoke contains a variety of oxidizing agents (Pryor, 1982), which induce lipid peroxidation. This results in oxidative cellular damage (Halliwell, 1987; Rahman and MacNee, 1996) and is believed to contribute to the pathogenesis of germ cell apoptosis. In agreement with these findings is the mounting evidence that the concentrations of natural antioxidants retinol and a-tocopherol in the blood stream are considerably reduced by cigarette smoking (Bridges et al.,
1990; Faruque et al., 1995). Researchers from the National Institute of Health and Environmental Protection of the Netherlands assessed the relationship between smoking habits and dietary intake of antioxidant nutrients and their rich food sources. Men who smoked more than 20 cigarettes, had lower serum concentration of retinol and vitamin C than those who never smoked, in spite of high intake of food sources rich in antioxidants. This relationship was most dramatic for the male heavy smokers (Zondervan et al., 1996), as shown in the present study, demonstrating a dose-dependent effect. This effect can be explained by increased utilization of antioxidant vitamins during neutralization of phagocyte-derived free radicals (Maderazo et al., 1990). Several studies showed that supraphysiological concentrations of vitamin C, a-tocopherol and b-carotene can reduce lipid peroxidation induced by cigarette smoking (Allard et al., 1994). This was confirmed at the cellular level, where these vitamins neutralized extracellular hypochlorous acid generated by activated neutrophils in vitro (Anderson et al., 1990). Cigarette smoking is a common habit for men attending an infertility clinic in our community with an adverse effect on sperm parameters (Omu et al., 1998). The association between low serum concentrations of retinol and a-tocopherol and low seminal concentrations of a-tocopherol and sperm dysfunction has important clinical implications in infertility. A previous study demonstrated low total antioxidant activity, retinol and a-tocopherol in men with seminal infections (Omu et al., 1998). In men with prostatitis and vesiculitis, defective spermatozoa and leukocytes generate large amounts of ROS (Aitken et al., 1992; Kessopoulou et al., 1992; Rajasekaran et al., 1995). Reduced forms of oxygen, such as superoxide radicals, hydroxyl radicals and hydrogen peroxide, are generated during aerobic metabolism. To counter the effects of ROS, cells possess antioxidants as scavengers of the free radicals. The peroxidative chain reaction of polyunsaturated lipids can be terminated by retinol and a-tocopherol, water-soluble antioxidants. Vitamin C recycles oxidized a-tocopherol (Chan, 1993; Coudary et al., 1995). With vitamin C, vitamin E has been shown to enhance cytokine production by the regeneration of its active form after it has RBMOnlineÒ
Article - Gender differences in retinol and a-tocopherol - MK Al-Azemi et al.
reacted with a free radical (Riley and Behrman, 1991). Menezo et al. (2007) evaluated the impact of antioxidant treatment on sperm DNA fragmentation and sperm head decondensation. Antioxidant treatment led to a decrease in sperm DNA fragmentation. However, it also led to an unexpected negative effect by increasing sperm decondensation. In an extensive review by Agarwal et al. (2004), the efficacy of antioxidants in male infertility was evaluated. Many clinical trials have shown the positive effects of antioxidants on male infertility; however, these studies have used different forms in different combinations and dosages for varying periods of time. Therefore, the evidence supporting antioxidant use does exist but a consensus is required regarding type and dosage of antioxidants to be used. Another interesting finding in the present study was the significantly reduced serum retinol and a-tocopherol concentrations in anovulatory women compared with those with ovulatory cycles. Most women having low serum retinol and a-tocopherol in the anovulatory group have polycystic ovary syndrome, with high circulating androgen, testosterone and LH:FSH ratio greater than 2. In a study conducted on postmenopausal women before and after hormone replacement therapy and 20 premenopausal women who served as controls, data showed that oestrogens do not reduce plasma a-tocopherol concentrations (Wu et al., 1996). The present study has demonstrated indirect evidence that women with high circulating androgen have lower serum retinol and a-tocopherol concentrations. Obesity is another factor associated with increased oxidative stress. Few studies have investigated the antioxidant system in obese patients with infertility. The findings in this study have demonstrated the effect of BMI on antioxidant status. Previous studies showed that obesity elevates oxidative stress by elevations in lipid peroxidation (malondialdehyde, hydroperoxides, 4-hydroxynonenal, isoprostanes, and conjugated dienes) or protein oxidation (8-hydroxydeoxyguanosine). Lipid peroxidation is associated with a low systemic antioxidant defence (i.e. antioxidant enzymes, tissue dietary antioxidants) as confirmed in this study. These findings are of clinical relevance in an infertility clinic setting. Obesity-related oxidative stress may be corrected by improving antioxidant defences through fat volume reduction via surgery, pharmacological agents, exercise and/or dietary modification such as vitamin A and E supplementation (Vincent et al., 2007). There are many dire long-term clinical implications of these findings from the perspective of infertility management. Men with sperm dysfunction and women with anovulation, especially if they are smokers and obese, could benefit from simple measures such as weight reduction programmes, smoking cessation and antioxidant supplements. The practice of actively treating these patients, who have an extreme desire for childbearing, without starting with such simple measures, may need revision. More research is certainly needed to further explore the association between the serum concentrations of retinol and a-tocopherol and infertilityrelated problems. Furthermore, there is a need to establish RBMOnlineÒ
the required preventive and therapeutic doses of these antioxidant nutrients in the management of such problems.
Acknowledgements The authors sincerely appreciate the contribution of Dr. Clifford Abiaka of Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences and Nursing, Kuwait University, in donating the standards used in the project.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 16 November 2008; refereed 17 December 2008; accepted 11 May 2009.
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