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doi:10.1093/humrep/den331 Human Reproduction Vol.23, No.12 pp. 2799–2805, 2008 Advance Access publication on August 28, 2008 Semen quality according to prenatal coffee and present caffeine exposure: two decades of follow-up of a pregnancy cohort C.H. Ramlau-Hansen1,4, A.M. Thulstrup1, J.P. Bonde1, J. Olsen2 and B.H. Bech3 1 Department of Occupational Medicine, Aarhus University Hospital, Norrebrogade 44, Building 2C, DK-8000 Aarhus C, Denmark; Department of Epidemiology, School of Public Health, UCLA, Los Angeles, CA 90095, USA; 3Department of Epidemiology, School of Public health, University of Aarhus 8000, Aarhus C, Denmark 2 4 Correspondence address. E-mail: [email protected] BACKGROUND: A few studies have investigated the association between male caffeine consumption in adult life and semen quality with conflicting results, but so far no studies have explored the effect of prenatal coffee exposure. We studied the association between prenatal coffee and current caffeine exposure and semen quality and levels of reproductive hormones. METHODS: From a Danish pregnancy cohort established in 1984–1987, 347 sons out of 5109 were selected for a follow-up study conducted 2005–2006. Semen and blood samples were analyzed for conventional semen characteristics and reproductive hormones and were related to information on maternal coffee consumption during pregnancy and present caffeine consumption. Data were available for 343 men. RESULTS: There was a tendency toward decreasing crude median semen volume (P 5 0.06) and adjusted mean testosterone (P 5 0.06) and inhibin B (P 5 0.09) concentrations with increasing maternal coffee consumption during pregnancy. Sons of mothers drinking 4–7 cups/day had lower testosterone levels than sons of mothers drinking 0–3 cups/day (P 5 0.04). Current male caffeine intake was associated with increasing testosterone levels (P 5 0.007). Men with a high caffeine intake had 14% higher concentration of testosterone than those with a low caffeine intake (P 5 0.008). CONCLUSIONS: The results observed in this study are only tentative, but they do not exclude a small to moderate effect of prenatal coffee exposure on semen volume and levels of reproductive hormones. Present adult caffeine intake did not show any clear associations with semen quality, but high caffeine intake was associated with a higher testosterone concentration. Keywords: caffeine; prenatal exposure; reproductive hormones; risk factor; sperm count Introduction Coffee consumption during pregnancy is high in Denmark, and about half of pregnant women drink coffee daily (Wisborg et al., 2003; Bech et al., 2005). Coffee is the main source of caffeine, a mild central system stimulant also found in tea, cola, cocoa and chocolate (Bunker and McWilliams, 1979). Caffeine crosses the placenta easily (Aldridge et al., 1981) and is found in both the fetus and in the newborn infant (Cazeneuve et al., 1994). Exposure to caffeine during pregnancy has, although not consistently, been associated with adverse pregnancy outcomes such as spontaneous abortion and stillbirth (Wisborg et al., 2003; Bech et al., 2005), reduced birthweight and intrauterine growth restriction (Grosso and Bracken, 2005). Whether prenatal caffeine exposure is associated with reduced semen quality has not been studied. However, a study on rats found that prenatal administration of theophylline (a caffeine metabolite) resulted in failure of development of seminiferous cords and, as a consequence, Leydig cells (Pollard et al., 2001). Caffeine concentrations in blood and semen are almost identical (Beach et al., 1984), and a few studies have assessed the association between current male coffee consumption and semen quality with conflicting results: the results from the cross-sectional studies of Oldereid et al. (1992) and Horak et al. (2003) suggest no association, whereas results from the studies of Adelusi et al. (1998), Marshburn et al. (1989) and Sobreiro et al. (2005) indicate that coffee consumption is associated with increased sperm motility. Vine et al. (1997) found weak evidence for an association between caffeine intake from coffee, tea and soft drinks and sperm nuclear morphometry, and Parazzini et al. (1993) found increasing risk of poor semen quality with increasing coffee consumption. These studies had limited confounder adjustment, but even if caffeine intake has no impact on current semen quality, exposure during organogenesis may impact gonadal development and thus later gonadal function. Coffee has been shown to be associated with low levels of estrogen (Petridou et al., 1992) and high levels of testosterone and sex hormone # The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] 2799 Ramlau-Hansen et al. binding globulin (Svartberg et al., 2003); all of which may affect growth of Sertoli cells during fetal life. Semen quality, in general, has possibly decreased during the past 60–70 years (Bostofte et al., 1983; Carlsen et al., 1992), and the number of couples seeking infertility treatment is increasing (Olsen et al., 1998). Therefore, it remains important to identify avoidable environmental risk factors for low semen quality. The aim of the present study was to investigate the association between prenatal coffee exposure and present caffeine exposure and semen quality and levels of reproductive hormones in a population-based study of young adult men. Materials and Methods Population The study participants were sons of mothers recruited to the Healthy Habits for Two cohort during their pregnancy from April 1984 to April 1987 (Olsen et al., 1989). The Healthy Habits for Two study took place in two Danish municipalities (Aalborg and Odense), and 11 980 women with singleton pregnancies (.80% of all invited) participated. They provided information on health related characteristics and lifestyle factors during pregnancy, including coffee and tea consumption, by filling out a self-administered questionnaire handed out by the midwives around the 36th week of gestation and returned in sealed envelopes to the university’s research department within a couple of weeks. Sons, who were alive and living in Denmark in December 2004, were identified in the Danish Civil Registration System (N ¼ 5109). Since the follow-up study was primarily designed to examine the association between prenatal smoking exposure and adult semen quality (Ramlau-Hansen et al., 2007), the participants were selected according to levels of maternal smoking during pregnancy, without knowing anything about their sons’ semen quality. Letters of invitation were sent consecutively, the oldest and those living close to either Aalborg (north of Jutland) or Odense (Funen) having priority, starting at the city centers and going as far as 30 km from the centers. Having a limited number of men heavily exposed to tobacco prenatally resulted in use of an expanded geographic area for this group. Additionally, men whose mothers had reported information on health-related issues from childhood to adolescence by means of a self-administered questionnaire when the sons were 16 – 19 years of age were given priority. A total of 716 men were invited to take part in the study, and 347 (48.5%) gave consent and participated. Of 100 men who declined participation by mail or telephone, 82 provided some information on their health. Information on maternal coffee and tea consumption during pregnancy was provided by mothers late in pregnancy, and information on coffee and cola consumption at the time of sampling was provided by the sons. Prenatal and present exposure information was available for 343 (98.8%) sons, who formed the study group for this study. The selected participants were 18–21 years of age and received an economic compensation for responding to the questionnaires and delivering a semen and a blood sample. Men with severe handicaps or congenital syndromes, such as spastic paraplegia or Down’s syndrome, as well as men with metabolic diseases or psychiatric disorders, were not invited. The study was approved by the regional ethics committee (registration number 20040174), and participation was conditional on written informed consent. Data collection Data were collected from February 2005 to January 2006. The participants were instructed to provide a semen sample by masturbating into 2800 a plastic container at home after at least 48 h of sexual abstinence. The container was then to be kept close to the body during transportation to the mobile laboratory to avoid cooling, and a single trained medical laboratory technician performed the initial semen analysis. Blood samples were taken between 7:25 a.m. and 7:15 p.m. (median time, 1:05 p.m.). The participants completed questionnaires on their reproductive experience, medical and lifestyle factors, including coffee and cola consumption, time and date of the preceding ejaculation, and spillage during sample collection. Exposure assessment The questions on coffee and tea consumption during pregnancy were phrased as follows: how many cups of coffee and how many cups of tea do you usually drink per day while being pregnant? (translated from Danish). The following response categories were given: 0–3 cups, 4– 7 cups and 8 or more cups. The main exposure in this study on prenatal exposure was daily coffee consumption, but in a subsequent analysis, the estimated total caffeine intake from coffee and tea was also used as the determinant: we estimated the caffeine content to be 100 mg in a cup of coffee, 50 mg in a cup of tea and 50 mg in half a liter of cola (Bunker and McWilliams, 1979), and arbitrary set the caffeine intake to 150, 550 and 950 mg for 0 –3 cups, 4–7 cups and 8 or more cups of coffee drunk per day, respectively. For tea, the caffeine intake was set to 75, 275 and 475 mg for 0–3 cups, 4–7 cups and 8 or more cups drunk per day, respectively. Then, we calculated the combined caffeine intake for each pregnant woman and divided the women into three groups according to daily caffeine intake during pregnancy: low caffeine, 225 mg (n ¼ 145), medium caffeine, 425–625 mg (n ¼ 143) and high caffeine, 825–1225 mg (n ¼ 47). However, the main determinant used in the analysis was cups of coffee per day. Among the sons, 50% did not drink coffee at all, and less than 20% drank coffee on a daily basis. Therefore, the combined caffeine intake from coffee and cola for each young man was used as the main determinant in the analysis on present exposure, even though the estimation of caffeine content in beverages such as coffee and tea is associated with substantial uncertainty (Bracken et al., 2002). The sons’ caffeine intake was estimated by adding caffeine from coffee and cola and dividing the sons into three groups: low caffeine, 0–25 mg (n ¼ 139), medium caffeine, 50 –125 mg (n ¼ 143) and high caffeine, 175–1075 mg (n ¼ 62). The questions to the sons was phrased: do you drink coffee/cola and if you do, how much? (translated from Danish). The following response categories were given for coffee (the arbitrary set caffeine content given in the parentheses was not disclosed in the questionnaire): none (0 mg), not daily (50 mg), 1– 4 cups/day (250 mg), 5–8 cups/day (650 mg) and more than 8 cups/day (1050 mg). In the same way, the following response categories (and arbitrary set caffeine content) were given for cola: none (0 mg), not daily/less than half a liter per day (25 mg), half a liter per day (50 mg), half to one liter per day (75 mg) and .1 l/ day (125 mg). Semen analysis Semen analyses were performed blinded to any prenatal conditions. Semen volume was estimated by its weight (1 g ¼ 1 ml). Sperm motility and sperm concentration were assessed as described in the World Health Organization’s WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (1999). Examination of 82.0% of the samples was initiated within 1 h, at which time it has been shown that the motility is stable (Makler et al., 1979), and examination of 99.7% of the samples was initiated within 2 h. Sperm morphology was determined using the strict criteria of Kruger et al. (1988). The laboratory took part in the European Caffeine and semen quality Society for Human Reproduction and Embryology Nordic external quality control program, and all control tests were within the limits set by this organization. Analysis of serum samples After centrifugation, serum was stored at 2808C for a maximum of 16 and a half months until analysis. Serum samples for total testosterone and follicle-stimulating hormone (FSH) were analyzed by Avida Centaur (Bayer Healthcare, Leverkusen, Germany). Inhibin B was measured by a commercially available enzyme-linked immunosorbent assay (Oxford Bio-Innovation Ltd, Oxford, UK) according to the manufacturer’s instructions. The blood samples were analyzed blinded to caffeine intake and as single measurements in a random order over a short period of time. The detection limits and total (intra- and interassay) coefficients of variation for the immunoassays were as follows: testosterone: 0.35–52.1 nmol/l, ,7.7%; FSH: 0.3–200 IU/l, ,4.0%; and inhibin B: 15.0–1000 pg/ml, ,7%. In two samples, the concentration of inhibin B was below the detections limit for the specific assay (15.0 pg/ml); therefore, the concentration was arbitrarily set to 14.0 pg/ml before statistical analyses were performed. The inhibin B samples were analyzed at the Laboratory of Reproductive Biology, University Hospital of Copenhagen, Denmark. Testosterone and FSH were analyzed at the Department of Clinical Chemistry, Aarhus University Hospital, Denmark. Statistical analysis Crude median, 25th and 75th percentiles were calculated for all outcome variables. For each of the outcome variables, we performed multiple linear regressions with prenatal coffee exposure (and caffeine in a subanalysis) and male adult caffeine consumption, respectively, as the main determinants. Low coffee/caffeine was considered the reference category. When we tested for trend, coffee/caffeine group was entered as a continuous explanatory variable, using low coffee/ caffeine as a starting point. Data on all outcome variables, with the exception of percentages of motile sperm and inhibin B, were cubic-root transformed to obtain an approximate normal distribution of residuals. Data on percentage of motile sperm were logit transformed. In this paper, back-transformed means are presented with 95% confidence intervals. Results were adjusted for season (summer/winter), history of diseases of the reproductive organs (cryptorchidism, hypospadias, varicocele, hydrocele, orchitis and chlamydia combined into one variable, present or not present), smoking (yes/no) and maternal smoking during pregnancy (yes/no). The semen outcome variables were additionally adjusted for abstinence time (48 h, 49 h–5 days, .5 days) and spillage during collection of the sample (yes, no). Data on participants who reported spillage during masturbation (n ¼ 88) were, however, excluded from all statistical analysis on semen volume and total sperm count. The results on motility were also adjusted for time from ejaculation to analysis (continuous, in minutes). In addition to season, diseases of the reproductive organs, and own or maternal smoking, the blood sample outcome variables were also adjusted for time of day of blood sampling (6:00– 8.59 a.m., 9:00 a.m. to 12:00 noon, after 12:00 noon). All statistics were performed by using Intercooled Stata 9 software (Stata Corporation, College Station, TX, USA). A two-tailed P-value of less than 0.05 was considered statistically significant. Results Maternal coffee consumption during pregnancy was associated with higher maternal age, smoking and lower family socioeconomic group (data not shown). Sons of mothers with a high coffee intake (.7 cups/day) had a lower birthweight and were more often smokers than sons of mothers with a low coffee intake (0 – 3 cups/day). Among sons of mothers with a high coffee intake, 25% drank themselves coffee on a daily basis compared with 15– 16% of sons of mothers with a lower intake (data not shown). There was a tendency toward a decreasing crude median semen volume and adjusted mean testosterone and inhibin B concentrations with increasing prenatal coffee exposure, as presented in Table I, but the trend tests were not statistically significant (P for trends respectively 0.06, 0.06 and 0.09). Sons of mothers drinking 4 – 7 cups/day had lower testosterone levels than sons of mothers drinking 0 – 3 cups/day (P ¼ 0.04). The crude median sperm concentration and total sperm count among sons of mothers consuming .7 cups of coffee per day were 33 million/ml and 101 million, which are, respectively, 21% and 28% lower than the crude medians among sons of mothers consuming 0 – 3 cups/day. The differences were, however, not statistically significant. Repeating the analyses using estimated prenatal caffeine exposure as the main determinant attenuated the associations (data not shown). No clear significant trends of decreasing or increasing semen quality or inhibin B or FSH levels with increasing present caffeine intake were found, as presented in Table II. However, increasing testosterone levels were associated with increasing caffeine intake (P ¼ 0.007). Men with a high caffeine intake had 14% higher concentration of testosterone than men with a low caffeine intake (P ¼ 0.008). We performed subanalyses for semen volume, sperm concentration and serum testosterone in which we additionally adjusted for maternal age (continuous, in years), maternal coffee/sons caffeine consumption (low, medium, high) and socioeconomic group (white-collar workers versus blue-collar workers, people in education or unemployed). We included one variable at a time, and the magnitude of effect of both prenatal coffee and present caffeine on the outcome variables was essentially unchanged by the adjustment. We also stratified the participants according to maternal smoking during pregnancy (yes/no) and diseases in the reproductive organs (present/not present) and repeated the analyses for semen volume, sperm concentration and testosterone with maternal coffee and sons’ caffeine consumption as the exposure (not at the same time). We found an indication that both stratifying variables modified the effect between maternal coffee intake during pregnancy and sperm concentration in the sons (P ¼ 0.07 and 0.04, respectively), and diseases in the reproductive organs modified the effect between sons caffeine intake and semen volume (P ¼ 0.03). However, the numbers in each stratum were small, and the direction of association was not clear (data not shown). Discussion The results from this first population-based follow-up study on prenatal caffeine exposure indicate that there may be an adverse effect of prenatal coffee exposure on semen quality and level of reproductive hormones in the sons, but the study 2801 Ramlau-Hansen et al. Table I. Semen and blood characteristics for 343 Danish young men stratified according to maternal coffee intake during pregnancy. Parameter Maternal coffee intake during pregnancy 0 –3 cups/day (n ¼ 208) Sperm concentration (millions/ml) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Semen volume (ml) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Sperm total count (millions) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Percent normal morphology sperm Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Percent motile sperm Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Testosterone (nmol/l) Median (p25– 75) Adjusted back-transformed mean (95% CI)§ FSH (IU/l) Median (p25– 75) Adjusted back-transformed mean (95% CI)§ Inhibin B (ng/ml) Median (p25– 75) Adjusted mean (95% CI)§ 4 –7 cups/day (n ¼ 91) Test for trend* .7 cups/day (n ¼ 44) P-value 42 (21, 80) 56 (43, 72) 39 (21, 89) 57 (41, 77) 33 (16, 102) 56 (36, 83) 0.62 1.00 3.1 (2.2, 4.0) 3.6 (3.2, 4.1) 2.7 (2.0, 3.6) 3.5 (3.0, 4.1) 2.5 (2.0, 3.7) 3.2 (2.6, 4.0) 0.06 0.19 140 (58, 271) 196 (146, 257) 103 (36, 289) 189 (129, 265) 101 (51, 282) 204 (123, 313) 0.38 0.96 5.0 (3.0, 8.0) 5.1 (4.0, 6.3) 6.0 (3.0, 9.0) 5.9 (4.4, 7.6) 5.0 (3.0, 7.0) 4.8 (3.2, 7.0) 0.68 0.75 71 (61, 76) 70 (63, 77) 69 (62, 77) 72 (63, 79) 68 (55, 78) 64 (53, 74) 0.89 0.31 16.7 (13.7, 20.2) 19.2 (17.4, 21.0) 16.2 (12.6, 19.5) 17.7 (15.7, 19.8) 17.2 (14.0, 20.3) 17.8 (15.4, 20.5) 0.43 0.06 2.9 (2.1, 4.4) 3.1 (2.5, 3.8) 3.0 (2.1, 4.0) 3.1 (2.4, 3.9) 2.9 (2.2, 4.3) 3.3 (2.4, 4.3) 0.94 0.75 154 (113, 185) 181 (161, 200) 150 (120, 180) 173 (150, 196) 141 (104, 177) 162 (133, 191) 0.20 0.09 SD, standard deviation; p, percentile; CI, confidence interval. *Trends were tested by Spearman’s rank correlation test (medians) and multiple linear regression (means), with 0– 3 cups of coffee per day as the starting point. §Back-transformed means were adjusted for season (summer/winter), history of diseases of the reproductive organs (present, not present), smoking (yes/no), and maternal smoking during pregnancy (yes/no). The semen outcome variables were additionally adjusted for abstinence time (48 h, 49 h– 5 days, .5 days) and spillage during collection of the sample (yes, no). Participants who reported spillage during collection (n ¼ 88) were excluded from all statistical analysis of semen volume and total sperm count. The results for motility were also adjusted for minutes from ejaculation to analysis (continuous). The blood sample outcome variables were additionally adjusted for time of day of blood sampling (6:00–8.59 a.m., 9:00 a.m.– 12:00 noon, or later than 12:00 noon). Sampling between October and March, no history of diseases of the reproductive organs, no smoking, no maternal smoking during pregnancy, 48 h –5 days of abstinence, no spillage, and blood sampling between 9:00 a.m. and 12:00 noon were chosen as the reference categories. had limited power. If real, the tendencies of decreasing concentrations of testosterone and inhibin B are compatible with effects of prenatal exposure on both the Sertoli and Leydig cell development. However, chance findings are not unlikely. On the other hand, present male caffeine exposure did not seem to affect adversely the semen quality or the levels of inhibin B or FSH in the cross-sectional part of this study. We did not find an association between caffeine and sperm motility, as earlier reported (Marshburn et al., 1989; Adelusi et al., 1998; Sobreiro et al., 2005), or an association between caffeine intake and sperm morphology, as suggested in one former study (Vine et al., 1997). In our study, participants were younger (18 – 21 years of age) and the study is population based and not based on infertility clinic patients, as in some of the former studies. The sample size is small for those with high exposure (n ¼ 62). In our study, testosterone was positively associated with present caffeine intake, which has also been reported by others (Ferrini and Barrett-Connor, 1998; Svartberg et al., 2003) and is also found in male animals (Pollard, 1988; Ezzat and el-Gohary, 1994). The animal studies suggest a stress-like pattern of endocrine response to the caffeine exposure (Pollard, 1988) and indicates that the association is from caffeine to testosterone and not that testosterone induces coffee intake (reverse causation). 2802 The mechanism behind the possible harmful effect of caffeine is not known. In both fetal and adult life, caffeine may act indirectly by impacting the hypothalamo-pituitarygonadal-system or by a direct toxic effect on the germinative epithelium (Eteng et al., 1997). Several studies have found an association between female caffeine (coffee alone or tea and caffeinated beverages included) intake and subfecundity (Wilcox et al., 1988; Olsen, 1991; Hatch and Bracken, 1993; Grodstein et al., 1993; Florack et al., 1994; Stanton and Gray, 1995; Bolumar et al., 1997; Jensen et al., 1998; Hassan and Killick, 2004), some mainly or only among smokers (Olsen, 1991; Bolumar et al., 1997). Semen quality and fecundity are correlated at sperm concentrations ,40 million/ml (Bonde et al., 1998) and the few studies evaluating the association between male caffeine intake and fecundity find increasing subfecundity with increasing or high male coffee/caffeine intake (Florack et al., 1994; Jensen et al., 1998; Hassan and Killick, 2004). Strengths of our follow-up study on the prenatal coffee/ caffeine exposure include the use of data on coffee and tea intake reported by the mother, when she was pregnant with the son we studied, so recall of consumption should be good and without differential recall bias. Information on usual coffee and tea consumption while being pregnant was provided by the mother late in third trimester and may reflect third Caffeine and semen quality Table II. Semen and blood characteristics for 343 Danish young men stratified according to present male caffeine intake (cola and coffee). Parameter Present caffeine intake Low caffeine (n ¼ 139) Sperm concentration (millions/ml) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Semen volume (ml) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Sperm total count (millions) Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Percent normal morphology sperm Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Percent motile sperm Median (p25, 75) Adjusted back-transformed mean (95% CI)§ Testosterone (nmol/l) Median (p25– 75) Adjusted back-transformed mean (95% CI)§ FSH (IU/l) Median (p25– 75) Adjusted back-transformed mean (95% CI)§ Inhibin B (ng/ml) Median (p25– 75) Adjusted mean (95% CI)§ Test for trend* Medium caffeine (n ¼ 143) High caffeine (n ¼ 62) P-value 34 (18, 78) 53 (40, 70) 44 (22, 90) 58 (43, 75) 44 (21, 96) 59 (42, 81) 0.22 0.45 2.8 (2.3, 3.8) 3.7 (3.2, 4.2) 3.3 (2.1, 4.1) 3.6 (3.2, 4.2) 2.5 (2.2, 3.7) 3.5 (2.9, 4.1) 0.69 0.47 118 (50, 206) 187 (135, 251) 113 (39, 288) 193 (139, 260) 145 (74, 351) 232 (158, 325) 0.19 0.27 5.5 (3.0, 8.5) 5.0 (3.9, 6.4) 5.0 (3.0, 8.0) 4.8 (3.7, 6.2) 6.8 (4.0, 10.0) 5.8 (4.2, 7.6) 0.50 0.48 69 (60, 76) 69 (61, 76) 69 (63, 77) 73 (65, 80) 71 (60, 77) 68 (59, 76) 0.22 0.84 16.2 (12.0, 19.4) 17.9 (16.1, 19.9) 16.8 (14.1, 20.1) 19.0 (17.2, 21.0) 18.4 (14.6, 21.7) 20.4 (18.1, 22.9) 0.003 0.007 2.9 (2.2, 4.4) 3.1 (2.5, 3.8) 2.8 (2.1, 3.7) 3.0 (2.4, 3.6) 3.2 (2.1, 4.8) 3.4 (2.7, 4.3) 0.74 0.53 144 (110, 180) 179 (158, 201) 160 (118, 185) 184 (163, 204) 153 (120, 187) 179 (154, 203) 0.20 0.92 SD, standard deviation; p, percentile; CI, confidence interval. *Trends were tested by Spearman’s rank correlation test (medians) and multiple linear regression (means), with low caffeine as the starting point. §Back-transformed means were adjusted for season (summer/winter), history of diseases of the reproductive organs (present, not present), smoking (yes/no), and maternal smoking during pregnancy (yes/no). The semen outcome variables were additionally adjusted for abstinence time (48 h, 49 h–5 days, .5 days) and spillage during collection of the sample (yes, no). Participants who reported spillage during collection (n ¼ 88) were excluded from all statistical analysis of semen volume and total sperm count. The results for motility were also adjusted for minutes from ejaculation to analysis (continuous). The blood sample outcome variables were additionally adjusted for time of day of blood sampling (6:00–8.59 a.m., 9:00 a.m. –12:00 noon or later than 12:00 noon). Sampling between October and March, no history of diseases of the reproductive organs, no smoking, no maternal smoking during pregnancy, 48 h– 5 days of abstinence, no spillage, and blood sampling between 9:00 a.m. and 12:00 noon were chosen as the reference categories. trimester rather than first trimester intake, although we asked for average intake. Since exposure early in pregnancy is more relevant with regard to development of the testicles, misclassification of exposure is possible. However, the percentage of mothers reporting the same consumption in pregnancy as before pregnancy accounted for, respectively, 74% among women drinking 0 – 3 cups of coffee per day, 77% among women drinking 4 – 7 cups of coffee per day and 89% among women drinking .7 cups of coffee per day. We also expect any misclassification in the cross-sectional study on present caffeine exposure reported by the male participants to be non-differential, since sons were not informed about the hypothesis we examined in the study. We have no information on maternal consumption of chocolate during pregnancy. We do, however, believe that the contribution of caffeine from chocolate accounts for only a small fraction of the total intake of caffeine. Caffeine intake from chocolate bars has been estimated to account for only 1.7% relative to caffeine from coffee (JP Bonde, Department of Occupational Medicine, Aarhus University Hospital, personal communication, 2008) in Danish women pregnant in 1990s (Jensen et al., 1998). Information on intake of chocolate and tea of the sons was also not available, but any information bias is most likely non-differential and therefore most likely leads to bias toward the null. We found that the associations attenuated, when we used the estimated prenatal caffeine (from coffee and tea) exposure as the main determinant instead of prenatal coffee exposure alone, perhaps because estimation of caffeine content is associated with substantial uncertainty (Bracken et al., 2002) or because there is another component only contained in coffee, and not caffeine or its metabolites, that in fetal life may has a programming effect on semen quality in sons. Our study’s participation rate (48.5%) was high for semen quality studies but not high enough to eliminate the risk of selection bias. To cause bias away from the null, selection has to be related to both semen quality and maternal/male caffeine intake, and the participation rate of men with both poor semen quality and a high caffeine exposure (maternal and own) intake must be higher. The source population was young, most had no reproductive experience, and they were not aware of the hypothesis evaluated, but we cannot exclude the possibility that the participants, despite their age and lack of reproductive experience may have been able to self-select themselves for the study in a way that caused selection bias. Studies have shown that older men with reduced fertility are more willing than other men to participate in semen-quality studies (Bonde et al., 1996), but it is unlikely that the sons in our study knew their mothers’ intake of coffee and tea during pregnancy or anything about their own semen quality. 2803 Ramlau-Hansen et al. We compared maternal coffee consumption between participants and invited non-participants (n ¼ 369) and found no difference. We also compared participants and nonparticipants who completed a small questionnaire on health (n ¼ 82) and found no difference in the proportion of men with diseases of the reproductive organs (including cryptorchidism and hypospadias). Semen data have a large variation and is associated with statistical uncertainty and misclassification. The misclassification is most likely random and may have attenuated the associations to a level where they cannot be detected. In our analysis, we controlled for abstinence time, diseases of the reproductive organs, time of day of blood sampling, maternal smoking during pregnancy and a number of other potential confounders, but residual confounding or confounding by other unknown factors cannot be ruled out. In the subanalyses, we controlled for maternal age, mothers’ coffee or sons’ caffeine intake and socioeconomic group, but doing so did not change the estimates. The results observed in this study are only tentative, but they do not exclude a small to moderate effect of prenatal coffee exposure on semen volume and levels of reproductive hormones. Present adult caffeine intake did not show any clear associations with semen quality. Men with a high caffeine intake had 14% higher concentration of testosterone than men with a low caffeine intake. Funding The study was supported by the Danish Medical Research Council (grant number 271-07-0051). 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