1 Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark.
2 Department of Environmental Medicine, University of Southern Denmark, Odense, Denmark.
3 Clinics of Surgery, Tartu University Clinicum, Tartu, Estonia.
4 Department of Obstetrics and Gynecology, Rikshospitalet, Oslo, Norway.
5 Institute of Biomedicine, Departments of Anatomy, Physiology and Paediatrics, University of Turku, Turku, Finland.
6 Institute of Endocrinology, Kaunas University of Medicine, Kaunas, Lithuania.
7 Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark.
Received for publication November 8, 2002; accepted for publication June 18, 2003.
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ABSTRACT |
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pregnancy; prenatal exposure delayed effects; semen; smoking; spermatozoa; sperm count
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INTRODUCTION |
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Because of the military draft system in Denmark, Finland, Norway, Lithuania, and Estonia, all young men are required to undergo a medical examination for determination of their fitness for military service. Therefore, these young men are from the general population and have not been selected with respect to their fertility status. Most men undergo the examination at 1820 years of age, but ages at examination vary. Collaboration with the military health board in each country provided us with a unique opportunity to invite these young men to participate in a study of male reproductive health. Participants answered a questionnaire about reproductive and general health and lifestyle factors, including in utero exposure to cigarette smoking. They provided blood samples for reproductive hormone measurements and semen samples for analysis. The aim of this study was to examine whether exposure to maternal smoking in utero or parental smoking in childhood influenced semen quality in adulthood.
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MATERIALS AND METHODS |
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The trained staff informed the men about the study and handed out written information. The men could fill in the informed consent form for participation and book an appointment immediately or return the consent form by mail. For a young man to participate in the study, he and his mother had to have been born and raised in the country where he was recruited. Participants received economic compensation for their travel expenses and/or lost working hours, according to local traditions. The participation rates at the study centers were 19 percent in Denmark (n = 889), 17 percent in Norway (n = 221), 13 percent in Finland (n = 313), 14 percent in Lithuania (n = 157), and 19 percent in Estonia (n = 190).
Ethical approval was obtained by local ethical committees in the participating countries. The procedures used were in accordance with the Helsinki Declaration of 1975, as revised in 1983. A detailed description of the semen analysis study has been given elsewhere (31).
Semen analysis
Each man provided a semen sample by masturbating into a wide-mouthed plastic container in a room close to the semen laboratory. The period of abstinence prior to sampling was recorded, and the semen sample was analyzed according to the World Health Organizations 1992 guidelines (32). A smear was taken from all semen samples and stained and preserved. A single physician in Finland assessed morphology using strict criteria (33). The investigator, who was blinded, evaluated 86 percent of the morphology smears. The remaining smears either were damaged in transport or had been fixated insufficiently.
An external quality control program was conducted throughout the study, as described previously (31). A previous study found interobserver variability in semen analysis between different laboratories (34), especially in motility assessment. However, the percentage of progressively moving sperm cells can be used as an outcome measure. The following semen variables were used as outcome variables: semen volume (ml), sperm concentration (millions/ml), percentage of motile sperm cells, percentage of sperm cells with normal morphology, and calculated total sperm count (concentration x volume, in millions).
Physical examination
All participants were examined by a physician. Testicular volumes were determined using a Prader orchidometer, and the mean of both testes was calculated. The possible presence of a varicocele, a hydrocele, or any genital malformation, the location of the testis in the scrotum, and the consistency of the testis and epididymis were recorded. Interobserver differences in testis size between the group members have previously been estimated (in a seminar where all physicians examined the same men) to be 16 percent (35). In addition, weight and height were measured, and body mass index was calculated as weight in kilograms divided by squared height in meters.
Questionnaire
All participants completed a questionnaire that was either submitted to their home address or handed out to them on the day of the military examination. It was returned to the physician at the time of physical examination. The questionnaire included information on previous and/or current diseases and genital conditions, such as cryptorchidism, inguinal hernia, varicocele, epididymitis, gonorrhea, chlamydia, and surgery for torsion of the testis. The men were asked whether they were still in school and, if not, what their highest level of education was and what their current work situation was. They reported on their smoking and alcohol intake during the week before completion of the questionnaire. Smoking habits were reported as the average number of cigarettes, cigars, or pipes smoked per day. Total weekly alcohol intake (number of drinks) was calculated by summarizing beer, wine, and liquor intake. In addition, a section of the questionnaire concerned possible in utero exposures; the men were asked to fill in that section in collaboration with their mothers, if possible (we recorded whether or not the information was obtained from the mother). The men were asked about their birth weight and length and whether they had been born at term. In addition, they were asked whether their mother had smoked while she was pregnant with them and if their parents had smoked at home (answer categories: neither parent smoked, one smoked, or both smoked).
Statistics
Outcome variables included testis size, semen volume, sperm concentration, total sperm count, and percentage of spermatozoa with normal morphology or motility. Data on sperm concentration and total sperm count were not normally distributed, so the median and interquartile range (25th75th percentiles) were calculated for these variables. Data on percentages of morphologically normal and motile cells, semen volume, and testis size were all normally distributed, so the mean and standard deviation were calculated for these variables. Diseases of the reproductive organs found to affect any of the outcome variables separately were combined into one variable for each outcome (present or not present).
We compared semen quality and testis size with information obtained from the questionnaire and the physical examination. We then characterized differences in the outcome variables with regard to exposure to smoking in utero and in childhood (explanatory variables) to determine possible confounding factors. Finally, we performed a multiple linear regression analysis, taking into account confounders affecting outcome variables and differently distributed among men exposed to smoking in utero and in childhood. In utero and childhood exposure to smoking were entered into the model as dummy variables. Normally distributed outcome variables were entered directly as continuous variables in the linear multiple regression analysis, whereas sperm concentration and total sperm count were transformed by the use of the natural logarithm to obtain normality. Confounders were excluded stepwise if they were not statistically significant at the 10 percent level. The results are presented with 95 percent confidence intervals. We evaluated the fit of the regression models by testing the residuals for normality and by inspecting the residual plots.
Since birth weight might be an intermediary factor in the pathway between in utero exposure to smoke and reduced semen quality and testis size, we calculated the contribution of birth weight to reduced semen quality and testis size by dividing the standardized beta coefficients for in utero smoking exposure with and without birth weight (as a con-tinuous variable) in the model and subtracting this from 1.
No significant interaction between smoking exposure in utero and smoking exposure in childhood was found (a dummy variable for different exposure points was added to regression analyses and was not significant). Accordingly, no interaction terms were entered into the multiple logistic regression analyses, but smoking exposure in utero was entered as a confounder in the model when childhood exposure was the explanatory variable and vice versa.
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RESULTS |
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Table 1 summarizes information from the men exposed to smoking in utero or in childhood. Current smoking and smoking exposure in utero and in childhood were correlated. Linear regression was performed with semen quality and testis size as dependent variables and exposure in utero and in childhood as explanatory variables. Before adjustment, men exposed to smoking in utero and in childhood had a smaller testis size, a lower sperm concentration and total sperm count, and fewer motile and morphologically normal sperm cells than unexposed men (table 2). After the data were controlled for body mass index, study center, diseases of the reproductive organs related to sperm concentration or total sperm count, period of abstinence (transformed by the use of the natural logarithm), and, for total sperm count, age and season, men exposed to smoking in utero had reductions in sperm concentration and total sperm count of 20.1 percent (95 percent confidence interval (CI): 6.8, 33.5) and 24.5 percent (95 percent CI: 9.5, 39.5), respectively, compared with unexposed men. Men exposed to smoking in utero had a 1.15 ml (95 percent CI: 0.66, 1.64) smaller testis size after data were controlled for body mass index, study center, age, still being in school, and diseases related to size. Percentages of motile and morphologically normal sperm cells were 1.85 (95 percent CI: 0.46, 3.23) and 0.64 (95 percent CI: 0.02, 1.30) percentage points lower, respectively, among men exposed in utero than in unexposed men after control for study center, age, diseases of the reproductive organs related to motility or morphology, and, for motility, smoking. The finding of 0.64 percentage points fewer morphologically normal cells should be interpreted in relation to the mean portion of morphologically normal sperm cells in the entire sample, which was only 7.6 percent; thus, this represents a considerable reduction. Exposure to smoking in childhood had an insignificant effect on semen quality and testis size after adjustment for potential confounders, including in utero exposure to smoking (table 2).
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We evaluated the validity of the smoking exposure variable by calculating mean birth weights among men exposed to smoking in utero and men not exposed. In the 60 percent of men for whom information on birth weight was available, the mean birth weight among the exposed was 3,361 g 267 g less than that among the unexposed (3,628 g).
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DISCUSSION |
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We found no independent effect of exposure to smoking in childhood, but this information was only classified as whether one or two parents had smoked at home. Therefore, it was impossible for us to determine the exact magnitude and timing of this exposure.
An effect of birth weight was found only when in utero exposure to smoking was excluded from the model and vice versa. This may indicate that birth weight is an intermediate factor in the relation between in utero exposure to smoking and semen quality and testis size. Nevertheless, birth weight accounted for less than 25 percent of the reduction in semen quality and testis size. The factors causing low birth weight may also be involved in impairment of the gonads during development. Previous studies on fertility in children with low birth weight have been conflicting (37, 38). None of the previous investigators controlled for the effect of in utero exposure to smoking. Since children with low birth weight may be more likely to have cryptorchidism and a low body mass index (39), conditions that may cause reduced semen quality, we repeated the analyses after exclusion of men with these conditions. An effect of in utero exposure to smoking on semen quality was still present in this group, a finding that seems to be in line with the assumption that birth weight is an intermediary factor.
Studies have found that both male and female mouse fetuses exposed to polycyclic aromatic hydrocarbons, the major toxic compounds found in cigarette smoke, have reduced fertility in adulthood (40). Polycyclic aromatic hydrocarbons increase apoptosis in oocytes when administered in utero and in adulthood and may also operate in testicular germ cells (41, 42). One study found an increased time to pregnancy among men prenatally exposed to cigarette smoking, but another could not confirm this finding (2830). These reports raised the question of whether exposure of the human male fetus to tobacco components may reduce adult reproductive capacity irreversibly. To our knowledge, only one study has examined semen quality among men exposed to smoking in utero (27), and no effect on semen characteristics was found. However, in that study, the mothers of the men participated in a randomized clinical trial of diethylstilbestrol, which may itself reduce semen quality (43, 44). Numerous studies included in a meta-analysis (26) examined the effect of current smoking and found an effect on semen quality. This could not be confirmed in our study. In fact, prenatal smoking exposure was the strongest predictor of poor semen quality, and current smoking had no independent effect, even though smoking exposures in utero, in childhood, and in adulthood were strongly correlated. In addition, the effect of in utero exposure on semen quality and testis size was also found among nonsmoking men. Since previous studies have not taken in utero exposure to smoking into account, the reported relation between semen quality and current smoking may be caused by in utero exposure.
There were limitations in our study. Firstly, the participation rate was low (1319 percent), and this may have caused a selection bias of unknown direction and magnitude. However, the men had essentially no prior knowledge of their own fertility potential, and therefore this is unlikely to have affected their motivation to participate. Bias in relation to the reported associations between in utero smoking exposure and semen quality is only likely if in-utero-exposed men with poor semen quality were oversampled. This is unlikely, since in utero exposure to smoking is not an established risk factor for poor semen quality.
Secondly, the quality of the information on prenatal smoking exposure in this study may be questioned, since we relied on retrospectively collected data. When we asked the men whether they had obtained the information on in utero exposure directly from their mothers and included only those men who indicated this, we found the same results. In addition, the mean birth weight among men exposed in utero was 264 g less than that among the unexposed; this corresponds to the deficit in birth weight from smoking found in other studies (45), thus indicating that the validity of the data on smoking exposure was good. However, this only indicates that the smoking information was valid for the 60 percent of the men who had information on birth weight. Some mothers may have a selective memory about their smoking habits, but since they answered the questionnaire before knowing the outcome of their sons semen analysis, this is unlikely to have affected the results. Ninety percent of the men with birth weight information had information on smoking habits from their mothers, and this information was probably more valid than the rest. In the two Baltic countries, the proportion of mothers who smoked was very low (<6 percent), and thus smoking in these countries may have been underreported. Nevertheless, underreporting would have misclassified exposed men as unexposed and thus caused underestimation of the true association.
The association between prenatal smoking exposure and semen quality could be due to confounding by other prenatal factors related to smoking, such as alcohol intake, diet, or drug use. We did not collect information on these factors. Present exposures or parental exposures (including an effect of paternal smoking mediated through fathers sperm) could also have confounded the association. We collected information about a wide range of present exposures with a possible effect on semen quality, such as smoking, alcohol intake, and education, but none of these factors affected semen quality, and they were controlled for in the multiple regression analysis. We collected information about intake during the week before questioning, because this has been reported to be more reliable than average exposure, but it does not measure exposure during the entire period of semen production. In addition, variation in smoking habits from week to week is low. Semen quality may vary with season, and it is affected by period of abstinence (46). However, the majority (78 percent) of the semen samples in this study were provided during a short period from October to February, and since we controlled for differences in period of abstinence, these factors are unlikely to explain our findings. No seasonal variation was observed at one of the centers (Turku), where samples were equally distributed over different seasons (31).
Thirdly, it is well known that interobserver variability in semen analysis between different laboratories may be present (34), especially in motility assessment, and this may have influenced our findings. Variation in physical examination (in testis size) results between physicians from these centers has previously been reported (35). However, we adjusted for differences between centers in the multiple regression analyses, and the relation between in utero exposure to smoking and semen quality and testis size was found independently in each country (except for Lithuania, where less than 2 percent of the pregnant women smoked). In addition, the technicians were blinded to the exposure information, and the same physician assessed all morphology slides. An external quality control program conducted throughout the study monitored semen analysis techniques. Thus, between-laboratory and physician variation are unlikely explanations for our findings.
In conclusion, we found reduced semen quality and a smaller testis size among young men exposed to smoking in utero in comparison with unexposed men, but we detected no independent effect of exposure to passive smoking in childhood. We do not believe that selection bias can explain our findings. To our knowledge, this study is the first to have shown a correlation between semen quality, testis size, and in utero exposure to cigarette smoking. Clearly, these findings must be confirmed by other studies. However, it seems reasonable to advise pregnant women to avoid smoking, since it not only may cause adverse birth outcomes but also may have long-term implications for the reproductive health of the offspring.
Note added in proof: Since the submission of this paper, Storgaard et al. (47) have published results showing a 48 percent reduction in semen concentration among sons exposed to maternal smoking of more than 10 cigarettes per day.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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