Download Semen quality according to prenatal coffee and

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
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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
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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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Submitted on May 16, 2008; resubmitted on August 5, 2008; accepted on
August 6, 2008
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