Keywords: asthenozoospermia, endocrine disruptors, lifestyle, semen quality, sperm concentration, toxicants

Transcription

1 RBMOnline - Vol 15. No Reproductive BioMedicine Online; on web 20 August 2007 A prospective study was carried out on 1005 male volunteers residing in the province of Barcelona. Participants were asked to complete a questionnaire concerning their health status and lifestyle, and provided a semen sample for analysis. Sperm concentration and percentages of motile, and normal forms were evaluated. The effect of smoking, alcohol consumption, stress and previous disease on semen parameters was also evaluated. Normal parameters were found in 22% of volunteers and sperm counts and/or percentage motility below normal threshold values were found in 78%. Asthenozoospermia was detected in 62% and oligozoospermia in 17% of volunteers. No statistically significant association was found between semen quality and age, with the exception of a decrease in semen volume (P = 0.04) and progressive motility (P = 0.01). No statistically significant differences in semen parameters were found between smokers and non-smokers or between males who consumed alcohol versus those that did not. However, stress had a negative effect on sperm concentration. In conclusion, the prevalence of normal semen parameters in the study population evaluated was markedly lower than that reported for the general population (60 70%). Since sperm concentration did not appear to decrease with age, the decrease observed in the last decades may be related to exposure to environmental toxicants during the fetal period, as previously suggested. Keywords: asthenozoospermia, endocrine disruptors, lifestyle, semen quality, sperm concentration, toxicants The ongoing debate about the putative decrease in semen quality was started in the 1970s (Nelson and Bunge, 1974). Since then and until 1992, several groups have reported similar results but without establishing a clear aetiology (Smith et al., 1978; James, 1980; Leto and Frensilli, 1981; Bostofte et al., 1983; Osser et al., 1984; Osegbe et al., 1986; Bendvold, 1989; Bendvold et al., 1991). The debate was reactivated by Carlsen et al. (1992), who reported the results of a meta-analysis of 61 articles published between 1938 and 1991, in which sperm concentration in semen of apparently fertile males was evaluated. By comparing studies from different geographical areas, the authors concluded that the average sperm concentration had decreased by almost 50% worldwide between 1940 and This study elicited great debate and a re-evaluation of the subject. Subsequent publications criticized the use of the retrospective study design and the type of statistical approach used in this meta-analysis (Brake and Krause, 1992; Bromwich et al., 1994; Farrow, 1994; Olsen et al., 1995; Bahadur et al., 1996; Fisch and Goluboff, 1996; Becker and Berhane, 1997; Swan et al., 1997; Saidi et al., 1999). During the last 20 years, at least in developed countries, a decrease in fertility rates has been observed. Besides the role that socio-economic changes may have played in this decrease of fertility rates, there seems to be a general consensus that the decrease in semen quality may have contributed to this decrease in fertility rates. The publication of several studies in the last four decades has contributed to feed the controversy about this 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 putative decrease in semen quality. There are studies that found a significant decrease in semen quality (Nelson and Bunge, 1974; Leto and Frensilli, 1981; Bostofte et al., 1983; Bendvold, 1989; Carlsen et al., 1992; Auger et al., 1995; Adamopoulos et al., 1996; Irvine et al., 1996; Van Waeleghem et al., 1996, Andersen et al., 2000; Jorgensen et al., 2001, 2002) and those that did not find evidence for this decrease in semen quality (MacLeod and Wang, 1979; Suominen and Vierula, 1993; Fisch et al., 1996; Paulsen et al., 1996; Berling and Wölner-Hanssen, 1997; Andolz et al., 1999). This decrease in semen quality has been associated with an increasing incidence of anomalies of the male genital tract (Toppari et al., 1996). In addition, the incidence of testicular cancer has increased worldwide in the last 4 decades (Adami et al., 1994; McKiernan et al., 1999). An increase in congenital anomalies, such as cryptorchidy and hypospadias, has also been observed (Toppari et al., 1996). In an effort to find an aetiology for all these anomalies of the male genital tract, the focus has been placed on endocrine disruptors found in the environment and that could affect gonadal development during fetal life (Sharpe and Skakkebaek, 1993; Toppari et al., 1996), resulting in the so-called gonadal dysgenesis syndrome (Skakkebaek et al., 2001). If the decrease in semen quality was related to chronic exposure to environmental factors, one would expect an effect on seminal parameters as a function of exposure time. On the other hand, if it was related to exposure to these factors during fetal life, one would not expect a decrease in semen quality as a function of age. Rather, the male would be born with a congenital defect in the testis that would negatively affect the process of spermatogenesis and semen quality. These toxic factors would include infectious disease, consumption of alcohol and tobacco, and exposure to environmental toxicants. The main objectives of this study were: (i) to evaluate semen quality in volunteers residing in the province of Barcelona, according to the guidelines of the World Health Organization (WHO) criteria; (ii) to determine what percentage of these males have normal semen parameters according to the WHO criteria; and (iii) to investigate the effects of infectious disease, lifestyle, consumption of alcohol and tobacco and exposure to environmental toxicants in the workplace on semen quality. From 1 February to 19 December 2003, 1005 volunteers from the province of Barcelona (from both urban and rural populations) were enrolled in the study. The age of the men ranged between 18 and 65 years. The study was carried out under the auspices of the Spanish Society of Andrology (ASESA) and the Colegio Oficial de Médicos of Barcelona. A recruitment campaign to enrol patients was organized by the posting of cards and informative posters in different locations of the province of Barcelona. Also, a press conference was organized to disseminate through the media the objectives of the study and to raise awareness in the province of Barcelona of the significance of the study. A prospective study was carried out in 1005 volunteers residing in the province of Barcelona with ages ranging between 18 and 65 years (mean age of 33.5 years). The volunteer population had a composition of about 60% university students and 40% from the rural area of the province of Barcelona. The motivation for volunteer participation was solely based on the incentive of a semen analysis free of charge. These males were asked to complete a questionnaire concerning their health status and lifestyle. Each volunteer provided a semen sample. Men were classified in two groups: group A (males who were not trying to produce offspring; n = 814) and group B (males who were hoping to produce offspring; n = 191). These two groups were differentiated in order to eliminate a potential selection bias concerning the recruitment of volunteers who agreed to participate in the study because they were hoping to produce offspring and could have an infertility problem. Groups A and B had a similar composition in terms of the distribution of volunteers from urban and rural areas. Age, semen volume, sperm concentration, total sperm count, concentration and total motile sperm count, percentage motility, progressive and non-progressive motility, and percentage normal forms, were compared in these two groups. The effect of smoking, alcohol consumption, stress, ejaculation frequency and the effect of previous disease on sperm concentration and progressive motility was evaluated. In order to determine the potential relationship between age and semen parameters, several age bands were established: 18 30, and 45 years. The reason why these age bands were selected was because the majority of the volunteers were in the age bands of years, years and above 45 years. The number of volunteers in age bands between and was relatively low and, therefore, were placed in the corresponding age group. Volunteers were asked to complete a questionnaire themselves concerning their health status and lifestyle. This questionnaire,requested information on current disease, if any, infectious diseases of the genital and urinary tracts, lifestyle, consumption of drugs, tobacco and alcohol, education, profession and exposure to environmental toxicants. A total of 1058 volunteers were contacted. Of these, 53 failed to fill out the questionnaire and, therefore, were not included in the study. The remaining 1005 volunteers agreed to fill out the questionnaire and to provide a semen sample for analysis. However, some of these volunteers did not complete all of the questions in the questionnaire (e.g. questions concerning alcohol or tobacco consumption, stress or cryptorchidism), thus the total number of volunteers that answered questions concerning tobacco consumption was 972, alcohol consumption 967, stress 972 and cryptorchidism 963. Of the 1005 volunteers included in the study, 961 answered all questions. The questionnaire used in this study was a standard one and fully validated. A questionnaire was also provided to measure the presence of stress in the volunteers. The questionnaire used was a modified Spielberger State Anxiety Inventory (STAI) questionnaire which is widely used for assessing acute states of anxiety

3 (Spielberger et al., 1970) and the effect of stress on semen parameters (Clarke et al., 1999). Volunteers participating in this study were asked to provide a semen sample obtained by masturbation after an abstinence period of 3 to 7 days. The semen samples were collected into a wide-mouth, sterile, polypropylene container and delivered to the andrology laboratory within 1 hour after collection. Semen sample collection took place in a consecutive manner over a 3-month period. Semen analysis was performed by three well-trained technicians. In order to minimize variability between observers, an internal quality control system was in place and a fourth experienced technician also examined all these semen samples. The coefficient of variation between the four technicians was below 10%. In addition, during the study, the centre participated in an external quality control system for semen analysis with Centro de Estudio e Investigación de la Fertilidad (CEIFER, Granada) in collaboration with the European Society for Human Reproduction and Embryology and the Asociación para el Estudio de la Biología de la Reproducción (ASEBIR). A total of 1005 semen samples were evaluated according to the WHO guidelines for semen analysis (World Health Organization, 1999). Semen parameters evaluated included: ejaculate volume, sperm concentration, total sperm count, percentage motile sperm and percentage normal forms, according to WHO criteria. Aliquots of the semen samples were analysed within min after semen liquefaction. Semen volume was measured using a calibrated pipette. A 5-μl aliquot was added to a Makler chamber (Sefi-Medical Instruments Ltd, Haifa, Israel) and sperm visualized by phase contrast microscopy at 200 magnification using an Olympus microscope. Sperm motility was evaluated according to the WHO criteria, and motility grades a, b and c were recorded. Each parameter was measured 10 times. The intra-assay variability was 10% and all four technicians were blinded to the questionnaire results. A μl aliquot was used to produce a smear in order to determine the percentage of normal forms in the semen. The smears were dried at room temperature and stained with the Hemacolor kit (Merck, Germany). The percentage of normal forms was obtained according to WHO criteria (World Health Organization, 1999). A descriptive study of the variables was carried out by calculating the measures of central tendency for quantitative variables and absolute and relative frequencies for qualitative variables. Following the descriptive study, an analysis of the possible associations of interest was carried out. Chi-squared and Pearson tests were used to compare percentages. For comparison of means between groups, the Student s t-test and analysis of variance (ANOVA) were used. The Kolgomorov Smirnov test was used to confirm the normal distribution of quantitative variables. Otherwise, equivalent non-parametric tests were used: Mann Whitney U test and Kruskall Wallis test. A bivariate analysis was carried out using chi-squared and multivariate analysis with logistic regression with the objective of identifying potential factors associated with semen quality in the group of volunteers of the province of Barcelona. Measures of association were obtained between the study variable and potential risk factors. The odds ratio (OR) and confidence intervals (CI) that may affect semen quality were also determined. For statistical inference, the null hypothesis was rejected (no association) when the P-value associated with the tests was less than Statistical analyses were performed using a Statistics Package for Social Sciences (SPSS) program version 11.5 (SPSS, Chicago, IL, USA). Of the 1005 semen samples evaluated, 22% had normal semen parameters according to WHO criteria, and 78% had a sperm concentration and/or sperm motility and/or percentage normal forms below normal threshold values. The semen parameter values in volunteers with normal and abnormal semen parameters are shown in Table 1. The seminal anomaly most frequently observed was asthenozoospermia, affecting 62.3% of all volunteers. Oligozoospermia alone or in combination with asthenozoospermia was detected in 17% (Table 1). There were no statistically significant differences in the distribution of semen quality between groups A and B. The mean ± SD values for abstinence time were 3.6 ± 0.8 days. The mean ± SD values for semen volume were 2.8 ± 1.7 ml. There were no significant differences in sperm concentration as a function of age (Table 2). In contrast, there was a significant decrease in percentage sperm motility (P = 0.01) and semen volume (P = 0.04) (Table 2). These findings are in good agreement with the report by Eskenazi et al. (2003). The effect of tobacco smoking on sperm concentration and sperm motility was also evaluated. The results are shown in Table 3 (a) and (b). Of the 972 volunteers that were asked to fill the questionnaire, 24.8% recorded that they were smokers. No statistically significant differences were found between smokers and non-smokers. Among the smokers, 66.3% smoked more than 10 cigarettes per day. No statistically significant differences were found when comparing seminal parameters as a function of the number of cigarettes smoked: 10 cigarettes per day or >10 cigarettes per day (Table 3b). The effect of alcohol consumption on semen parameters was also evaluated. Of the 967 volunteers that filled the questionnaire, 54.5% reported alcohol consumption on a daily basis ( 10 g/day of ethanol). No statistically significant differences in semen parameters between drinkers and non-drinkers were found (Table 4). The effect of stress on sperm concentration and progressive motility was also evaluated (Table 5). Of the 972 volunteers that filled the questionnaire, 66.6% reported experiencing stress

4 Table 1. Semen parameters in a population of volunteers from the province of Barcelona. Parameter Abnormal semen parameters (n = 216) Normal semen parameters (n = 789) Total (n = 1005) P-value Mean SD 95% CI Range Mean SD 95% CI Range Mean SD 95% CI Range Age (years) Semen volume (ml) NS Concentration of <0.01 spermatozoa Total sperm <0.01 count (x 10 6 ) Motile sperm <0.01 Total motile <0.01 count (x 10 6 ) Motility (a + b < c) (%) Grade a (%) <0.01 Grade b (%) <0.01 Grades a + b (%) <0.01 Grade c (%) <0.01 Normal forms (%) <0.01 CI: confidence interval; NS: not statistically significant.

5 Table 2. Effect of age on semen parameters. Parameter Age n Mean SD Confidence interval (95%) Range P-value group Lower limit Upper limit Minimal Maximal (years) Concentration NS of spermatozoa Motility (%) Volume (ml) NS: not statistically significant. Table 3. Effect of smoking tobacco on semen parameters: (a) smokers versus non-smokers; (b) number of cigarettes smoked daily. (a) Parameter Non-smokers (n = 731) Smokers (n = 241) P-value Mean SD 95% CI Range Mean SD 95% CI Range Concentration NS of spermatozoa Motility grade a (%) NS Motility grades NS a + b (%) NS: not statistically significant. (b) Parameter 10 cigarettes/day (n = 120) >10 cigarettes/day (n = 121) P-value Mean SD 95% CI Range Mean SD 95% CI Range Concentration NS of spermatozoa Motility grade a (%) NS Motility grades NS a + b (%) NS: not statistically significant.

6 Table 4. Effect of alcohol consumption on semen parameters. Parameter No alcohol consumption Alcohol consumption ( 10 g P-value (n = 440) ethanol/day) (n = 527) Mean SD 95% CI Range Mean SD 95% CI Range Concentration of NS spermatozoa Motility grade a (%) NS Motility grades NS a + b (%) NS: not statistically significant. Table 5. Effect of stress on semen parameters. Parameter No stress (n = 324) Stress (n = 648) P-value Mean SD 95% CI Range Mean SD 95% CI Range Concentration of spermatozoa (x 10 6 /ml) Motility grade a (%) NS Motility grades NS a + b (%) NS: not statistically significant. of variable intensity (low 30.6%, moderate 30%, and high 6%). A statistically significant difference in sperm concentration was found (P = 0.004). Sperm concentration was 63.4 ± /ml in volunteers that reported no stress versus 57.1 ± 46.1 x 10 6 /ml (P = 0.04) in volunteers that admitted to having stress. No effect on percentage motility, grade a motility (14.3 ± 10.0% versus 14.7 ± 10.2%) or grades a + b motility (41.1 ± 19.5% versus 43.1 ± 18.3%) was found as a function of stress. No statistically significant differences were found as a function of stress intensity. The effect of previous disease on semen quality was also evaluated (Table 6). About 13% of the volunteers that filled in the questionnaire had at least one child. The most frequent reported disease was cryptorchidism (11.5%) followed by sexual transmitted diseases (5.3%). Only the presence of cryptorchidism in volunteers showed a statistically significant negative correlation with sperm concentration (P < 0.01) and sperm motility (grade a motility P = 0.02, grades a + b motility P = 0.01). The results of bivariate analysis are shown in Table 7 and Figures 1 5. These results indicate that there was a statistically significant association between semen quality and the risk factors stress and cryptochidism. There was no statistically significant association between semen quality and tobacco or alcohol consumption. A logistic regression model was established in the multivariate analysis where the variable considered was semen quality (normal/abnormal) and the explicative variables were stress and cryptorchidism (Table 8) with the OR and CI shown in Tables 7 and 8.

7 Table 6. Effect of cryptorchidism on seminal parameters. Parameter No cryptorchidism (n = 865) Cryptorchidism (n = 98) P-value Mean SD 95% CI Range Mean SD 95% CI Range Concentration <0.01 of spermatozoa Motility grade a (%) Motility grades a + b (%) Table 7. Comparison of semen parameters by bivariate analysis. Variable Detail Normal semen Abnormal semen Bivariate analysis parameters n (%) parameters n (%) (Chi-squared tests) P-value RR 95% CI Age (years) (38.1) 258 (32.8) NS (56.7) 479 (60.9) (5.1) 49 (6.2) 1.00 Tobacco Non-smokers 160 (75.5) 571 (75.1) NS Smokers 52 (24.5) 189 (24.9) Alcohol No alcohol consumption 99 (46.9) 341 (45.1) NS Alcohol consumption 112 (53.1) 415 (54.9) Stress No stress 88 (41.7) 236 (31.0) Stress 123 (58.3) 525 (69.0) Cryptorchidism No cryptorchidism 198 (94.7) 667 (88.5) Cryptorchidism 11 (5.3) 87 (11.5) CI = confidence interval; NS = not statistically significant; RR = relative risk. Figure 1. Semen parameters versus age groups. The Y-axis values represent the percentage of volunteers that corresponds to each age group. The group with the highest proportion of abnormal semen parameters was years of age and the group with the lowest proportion was years of age. This abnormality in semen parameters corresponded to progressive decrease in motility and semen volume but not in sperm concentration.

8 Figure 2. Semen parameters versus tobacco consumption. No statistically significant association was found between semen parameters and smoking. Figure 3. Semen parameters versus alcohol consumption. No statistically significant association was found between semen parameters and alcohol consumption. Figure 4. Semen parameters versus stress. The percentage of volunteers with abnormal semen parameters was higher in the group that reported stress. The specific semen parameter affected was sperm concentration. This difference was statistically significant (P = 0.04). Figure 5. Semen parameters versus cryptorchidism. The percentage of volunteers with abnormal semen parameters was higher in the group with cryptorchidism. This difference was statistically significant (P = 0.008). Table 8. Comparison of semen parameters by multivariate analysis. Variable B SE df P-value OR 95% CI Stress Cryptorchidism Constant B = equation coefficients; CI = confidence interval; df = degrees of freedom; OR = odds ratio; SE = standard error.

9 The results of this prospective cohort study indicate that the prevalence of normozoospermia in the population of volunteers from the province of Barcelona was 22%. This prevalence is significantly lower than that reported by Eskenazi et al. (2003) for the general population, which ranged between 60 and 70%. Although semen volume ranged between 0.2 and 13.6 ml, the average value was 2.6 ml, which is consistent with the volume found for the normal population. Concerning the effect of abstinence time on semen volume, although an abstinence time of 3 to 7 days was allowed, the mean abstinence time for the study population was 3.6 days. However, it cannot be ruled out that abstinence time could have had an effect on the results. In order to avoid potential bias in volunteer selection, with or without infertility problems, two groups were established, based on whether they were trying to produce offspring or not. Since no significant differences in seminal parameters were found between these two groups, it can be safely excluded as a selection bias. In addition, the fact that no statistically significant differences were found in the distribution of semen quality between these two groups further strengthens the validity of the results obtained with the combined data. Since semen sample collection took place in a consecutive manner over a 3-month period, the results obtained may not be representative of all seasons. The potential effect of obesity as a confounding variable cannot be ruled out since this condition was not evaluated in the study. Obesity is a major potential confounding variable that may affect semen parameters. A proportion of smokers are also obese and, unfortunately, continue to smoke heavily in the mistaken belief that smoking will cause weight loss (Carroll et al., 2006). Therefore, the observation of semen parameters in such smokers could also be due to their obesity. Obesity prevalence rates are known to be very high in industrialised areas like Barcelona in Spain and the West Midlands in England (World Health Organization, 1998; Department of Health, 2001; United States Public Health Service, 2001; Popkin, 2007). Tobacco and alcohol consumption did not appear to have a significant effect on semen quality. These results are consistent with those reported by Martini et al. (2004), where they also did not find differences in semen quality in males that consumed alcohol or tobacco and also with those reported by Dikshit et al. (1987). Said et al. (2005) reported a negative effect of tobacco on semen quality, although it was related to tobacco chewing and not to cigarette smoking. More recently, smoking has been reported as a risk factor for decreased semen quality (Ramlau- Hansen et al., 2007). The discrepancies in the results of these studies need to be further evaluated. Thorup et al. (2006) reported an association between maternal cigarette smoking during pregnancy, cryptorchidism and decreased semen quality. The results of this study indicate that the group of boys with cryptorchidism whose mothers had smoked heavily during pregnancy (i.e. more than 10 cigarettes daily throughout pregnancy) had a significantly increased risk of bilateral cryptorchidism (52%), and a decreased number of spermatogonia and gonocytes per tubule cross-section compared with boys whose mothers did not smoke. Although stress had a statistically significant negative effect on sperm concentration (P = 0.04), the difficulty in standardizing stress intensity in the volunteers did not allow us to draw definite conclusions from the results of the study. One apparent limitation of the study could be the fact that only one semen sample per volunteer was evaluated. However, since the number of volunteers evaluated was relatively high, this would tend to minimize the potential effect of sample variability of semen quality in the population studies. No significant differences in sperm concentration were found as a function of age or of chronic exposure to environmental factors, which suggests that the relatively low semen quality found in the study population could be related to exposure to toxic factors such as endocrine disruptors during the prenatal period. Endocrine disruptors could induce alterations in the germ cell line leading to alterations of meiosis, maturation block during spermatogenesis and a subsequent reduction in sperm concentration. If this damage were produced after puberty, one would expect to find a worsening of semen quality as a function of age. Recent studies show that there is no correlation between sperm concentration and male age (Eskenazi et al., 2003). This is also supported by the report of Carlsen et al. (2005) in which no statistically significant differences in semen quality were found in a group of 158 young Danish adolescents studied over a period of 4 years. A study reported by Zheng et al. (1997) concluded that the decrease in sperm concentration observed in the Danish population corresponds to males born after 1950 and not before, suggesting that after 1950 a significant increase in the exposure to toxic environmental factors could explain, at least in part, the observed decrease in semen quality. These results are in good agreement with those reported by Andersen et al. (2000) where a significant decrease in semen quality in a population of adolescents was found. These findings are consistent with an environmental effect during the prenatal period. However, these results do not rule out the possibility that the putative testicular damage induced by environmental toxicants, such as endocrine disruptors, would not occur during the post-natal period before adolescence. However, if that were the case, this would not explain the increase in the incidence of congenital testicular anomalies observed in recent decades. Exposure to endocrine disruptors during the fetal period would be correlated with a higher prevalence of congenital anomalies such as hypospady, testicular cancer and cryptorchidism, which are all manifestations of the so-called testicular dysgenesis syndrome (Boisen et al., 2001; Skakkebaek, 2002, 2003). Perhaps the decrease in sperm concentration could be another manifestation of this syndrome. The findings reported in these study, including the effect of lifestyle factors and stress on semen quality, are subject to the limitations already mentioned and, therefore, should be interpreted with caution. In conclusion, the prevalence of normal semen parameters found in the population of volunteers from the province of Barcelona was markedly lower than that reported for the general population (Eskenazi et al., 2003). The consumption of tobacco and alcohol did not have a significant effect on semen quality,

10 while stress did appear to have a negative effect on sperm concentration, although this effect needs to be confirmed in a quantitative manner. Studies are currently underway to compare the concentration of endocrine disruptors in women during pregnancy and lactation from populations with significant differences in semen quality. We would like to thank all the volunteers that participated in the study. We also would like to thank Professor Ashok Agarwal for critical review of this manuscript. Adami H-O, Bergström R, Möhner M et al Testicular cancer in nine Northern European countries. International Journal of Cancer 59, Adamopoulos DA, Pappa A, Nicopoulou S et al Seminal volume and total sperm number trends in men attending subfertility clinics in the Greater Athens area during the period Human Reproduction 11, Andersen AG, Jensen TK, Carlsen E et al High frequency of suboptimal semen quality in an unselected population of young men. 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