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Human semen quality in the new
millennium: a prospective cross-
sectional population-based study of 4867
men
Niels Jørgensen,
1
Ulla Nordstro
¨
m Joensen,
1
Tina Kold Jensen,
1
Martin Blomberg Jensen,
1
Kristian Almstrup,
1
Inge Ahlmann Olesen,
1
Anders Juul,
1
Anna-Maria Andersson,
1
Elisabeth Carlsen,
1,2
Jørgen Holm Petersen,
3
Jorma Toppari,
4,5
Niels E Skakkebæk
1
ABSTRACT
Objectives:
Considerable interest and controversy
over a possible decline in semen quality during the
20th century raised concern that semen quality could
have reached a critically low level where it might affect
human reproduction. The authors therefore initiated
a study to assess reproductive health in men from the
general population and to monitor changes in semen
quality over time.
Design: Cross-sectional study of men from the general
Danish population. Inclusion criteria were place of
residence in the Copenhagen area, and both the man
and his mother being born and raised in Denmark.
Men with severe or chronic diseases were not
included.
Setting: Danish one-centre study.
Participants: 4867 men, median age 19 years,
included from 1996 to 2010.
Outcome measures: Semen volume, sperm
concentration, total sperm count, sperm motility and
sperm morphology.
Results: Only 23% of participants had optimal sperm
concentration and sperm morphology. Comparing with
historic data of men attending a Copenhagen infertility
clinic in the 1940s and men who recently became
fathers, these two groups had significantly better
semen quality than our study group from the general
population. Over the 15 years, median sperm
concentration increased from 43 to 48 million/ml
(p¼0.02) and total sperm count from 132 to 151
million (p¼0.001). The median percentage of motile
spermatozoa and abnormal spermatozoa were 68%
and 93%, and did not change during the study period.
Conclusions: This large prospective study of semen
quality among young men of the general population
showed an increasing trend in sperm concentration
and total sperm count. However, only one in four men
had optimal semen quality. In addition, one in four will
most likely face a prolonged waiting time to pregnancy
if they in the future want to father a child and another
15% are at risk of the need of fertility treatment. Thus,
reduced semen quality seems so frequent that it may
impair the fertility rates and further increase the
demand for assisted reproduction.
To cite: Jørgensen N,
Joensen UN, Jensen TK,
et al. Human semen quality in
the new millennium:
a prospective cross-sectional
population-based study of
4867 men. BMJ Open
2012;2:e000990. doi:10.
1136/bmjopen-2012-000990
<
Prepublication history and
additional materials for this
paper are available online. To
view these files please visit
the journal online (http://dx.
doi.org/10.1136/
bmjopen-2012-000990).
Received 6 February 2012
Accepted 29 May 2012
This final article is available
for use under the terms of
the Creative Commons
Attribution Non-Commercial
2.0 Licence; see
http://bmjopen.bmj.com
For numbered affiliations see
end of article.
Correspondence to
Dr Niels Jørgensen; niels.
joergensen@rh.regionh.dk
ARTICLE SUMMARY
Article focus
-
A paper by Carlsen et al 20 years ago (BMJ
1992;305:609e13) raised controversy with
evidence of a decline in semen quality, and
several studies on semen quality in human
populations have followed.
-
There has been a lack of larger, prospectively
collected quality-controlled data on semen
quality in the general population.
Key messages
-
This study brings good and bad news.
-
Fifteen years monitoring of semen quality in men
of the general population indicated a slight
increase in both median sperm concentration
and total sperm count.
-
However, still only a fraction of the men (23%)
had optimal sperm concentration and sperm
morphology, and the median percentage of
abnormal spermatozoa was as high as 93%
with no sign of improvement during the study
period.
-
Approximately 15% of the men had a sperm
concentration at a level that would indicate a high
risk of needing future fertility treatment if they
want to father a child, and another 27% of the
men will be at risk of a prolonged waiting time to
pregnancy.
Strengths and limitations of this study
-
Large prospective study of semen quality among
men of the general population unselected with
regard to fertility.
-
Standardised inclusion and investigation proce-
dures.
-
Lack of historical, directly comparable data.
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 1
Open Access Research
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INTRODUCTION
In the 1990s, a meta-analysis by Carlsen et al,
1
showing
a decline in human semen qual ity, initiated a heat ed
scientific debate. The discussion has recently resurfaced
in three papers in Epidemiology
2e4
and a news article in
Science.
5
The Carlsen paper, which was a review and meta-
analysis of internationally published data on semen
quality among healthy m en, suggested that there had
been a decline in sperm concentration and total sperm
count over a period of 50 years. Many were sceptical
about the results, and this prompted several researchers
to study trends in their own countries, mostly based on
data from semen banks or semen donor registries. The
resulting papers reported heterogeneous findings
(reviewed in Jouannet et al
6
and Merzenich et al
7
), with
some confirming a decreasing trend in semen quality,
and others not. In 2000, an updated comprehensive
meta-analysis was undertaken by Swan et al
8
that
confirmed the downward trend. During the same
period, there is strong evidence for a worldwide increase
in the incidence of testicular germ cell cancer, a disease
linked to decreased semen quality.
9e11
The background for the interest and controversy over
trends in semen quality was the obvious concern that
semen quality could have reached a critically low level
where it might affect fecundity (ie, the ability to repro-
duce). Therefore, since 1996, we have carried out
a prospective quality-controlled study of semen quality in
annual cohorts of men from the general Danish popu-
lation. A total of 4867 individuals have been included in
this study.
METHODS
The study population was men from the general Danish
population from the Copenhagen area examined in
1996e2010. For interpretation of the results, we
compared them to published data of two other studies
from the Copenhagen area: a recent study of fertile men
(male partners of pregnant women) examined in
1996e1998 by our group
12
and historical data of male
partners from infertile couples examined in 1939e1943
by Dr Richard Hammen.
13
Study population: men from the general population
examined in 1996e2010
In Denmark, all men, except those with severe or
ch
ronic diseases (<15%), are required to attend
a medical examination before being considered for
military service. Men are called upon to present them-
selves at th e age of 18e19 years, but some postpone this
examination until complet ion of their education. Men
attending the medical examinations are therefore
considered representative of the general population of
young men.
In collaboration with the military health authority,
men attending these medical examinations in the
greater Copenhagen area of Denmark were asked to
participate in the pre sent study, irrespective of whether
they were declared fit for military service or not. Further
inclusion criteria for this publication were: place of
residence in the greater Copenhagen area and both the
men and their mothers being born and raised in
Denmark. Those men who consented to participate were
given an appointment for examination at the Depart-
ment of Growth and Reproduction at Rigshospitalet
(Copenhagen, Denmark). Participants were instructed
to abstain from ejaculation for at least 48 h prior to
attendance at Rigshospitalet, where each man returned
a completed questionnaire, underwent a physical
examination and provided a semen sample. Participants
received a financial compensation (approximately V65).
Participants in this ongoing study have been included
since September 1996. Two of our previous publications
have directly focused on the semen quality level of men
examined from September 1996 to March 1998.
14 15
Other
publications have included information based on
subpopulations of men examined until the end of
2007,
16e35
but no previous trend analysis has been
performed on the material from the entire period.
Participants examined up until the end of December 2010
were included in the present publication, with 4901 men
fulfilling the inclusion criteria. The participation rate
among invited men ranged from 19% to 31%, with an
overall average of 24%. Data from 34 of the men were
excluded: 27 with previous or current use of anabolic
steroids, six who had previously received chemotherapy
for a malignant disease and one man who failed to deliver
a semen sample (he was later diagnosed with testosterone
deficiency due to a 46, XX-male karyotype). Thus, results
from the remaining 4867 men are reported here. The
study comprised annual cohorts of 240e543 men (median
276), 18e29 years of age (median 19). A detailed
description of the study population based on question-
naire information and results from the physical examina-
tion (see below) is summarised in table 1. The three types
of information is presented in table 1, ‘Been diagnosed as
having’, ‘Been treated for’ and ‘Has’ are based on ques-
tions phrased as ‘Has a doctor ever diagnosed you as
having.’, ‘Have you ever been treated for.’ and ‘Have
you ever.’, respectively. Within 3 months prior to partic-
ipation, 601 men (12%) had used medication, mainly
antibiotics, painkillers or asthma/allergy medicine.
Questionnaires
A standardised questionnaire was developed for this
study. In order to ensure the quality of the information
regarding previous conditions, the questionnaire was
sent to participants before their attendance at the
hospital, and they were asked to complete it beforehand
anddif possibledin collaboration with their parents.
The questionnaire included information on previous or
current diseases, including any known history of fertility
potential, and some lifestyle factors. The questionnaire
has been revised during the course of the study, mainly
with new questions being added, and the current
publication includes relevant information available from
all participants throughout the study.
2
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990
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Table 1 Physical appearance and self-reported information of young men from the general population in the Copenhagen area, Denmark
Investigation period
1996e2010, total (N[4867) 1996e2000 (N[1339) 2001e2005 (N[2254) 2006e2010 (N[1274)
Difference
between
the three
groups,
p Value
Mean (SD) Median (5e95) Mean (SD) Median (5e95) Mean (SD) Median (5e95) Mean (SD) Median (5e95)
Physical appearance
Age (years)* 19.4 (1.2) 19.0 (18.4e21.7) 19.6 (1.4) 19.0 (18.5e22.4) 19.3 (1.1) 18.9 (18.4e21.3) 19.4 (1.2) 19.0 (18.4e21.8) <0.0005{{
Height (m) 1.81 (0.06) 1.81 (1.71e 1.92) 1.81 (0.07) 1.81 (1.71e1.92) 1.81 (0.07) 1.81 (1.70e1.91) 1.82 (0.06) 1.82 (1.71e1.93) <0.0005{{
Weight (kg) 75.1 (11.5) 73.5 (59.4e 96.4) 75.2 (11.7) 73.6 (59.0e97.5) 74.9 (11.5) 73.3 (59.2e97.0) 75.5 (11.2) 74.1 (60.1e96.0) 0.15{{
BMI (kg/m
2
) 22.9 (3.1) 22.4 (18.7e28.8) 22.9 (3.2) 22.4 (18.8e28.9) 22.9 (3.2) 22.4 (18.7e29.0) 22.8 (3.1) 22.4 (18.7e25.6) 0.8{{
Testis size (ml)y 20 (5) 20 (13e28) 20 (5) 20 (12e 28) 20 (5) 20 (13e 28) 22 (5) 23 (14e 29) <0.0005{{
Testis size (ml), US 15 (4) 14 (9 e 22) 15 (5) 15 (9 e 24) 14 (4) 14 (9 e22) 14 (4) 14 (9 e21) <0.0005{{
Lifestyle
Cigarettes daily,
all men
4.1 (6.7) 0.0 (0.0e20.0) 5.0 (7.4) 0.0 (0.0e20.0) 3.9 (6.5) 0.0 (0.0e 20.0) 3.7 (6.2) 0.0 (0.0e18.0) 0.004{{
Cigarettes daily,
smokers only
9.9 (7.1) 10.0 (1.0e20.0) 11.7 (7.1) 10.0 (1.0e20.0) 9.9 (6.9) 10.0 (1.0e20.0) 8.1 (6.9) 7.0 (0.1e 20.0) <0.0005{{
Alcohol consumption
(units)z
14 (14) 11 (0 e 40) 13 (13) 11 (0 e 38) 14 (14) 11 (0 e37) 15 (16) 12 (0 e42) 0.1{{
Ejaculation
abstinence (hours)
81 (117) 63 (37e155) 86 (95) 63 (35e 168) 81 (137) 62 (38e 135) 77 (96) 63 (37e134) 0.1{{
%%%%
Taken medicationx 12.5 14.6 9.7 15.1 <0.0005{{
Smoker 41.7 42.5 39.1 45.4 0.001{{
Previous smoker 3.1 2.0 2.3 5.8 <0.0005{{
Never-smoker 55.2 55.6 58.6 48.7 <0.0005{{
Mother
smoked in
pregnancy
38.0 36.2 38.3 29.1 <0.0005{{
Been diagnosed as
having
Varicocele 0.6 0.5 0.7 0.4 0.4***
Epididymitis 0.3 0.3 0.5 0.2 0.3***
Sexual transmitted
disease{
4.3 2.2 4.6 6.2 <0.0005***
Cystitis 2.4 1.4 2.4 3.6 0.002***
Diabetes 0.02 0.0 0.04 0.0 0.6***
Thyroid disease 0.04 0.0 0.05 0.08 0.001***
Continued
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 3
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Table 1 Continued
Investigation period
1996e2010,
total (N[4867) 1996e2000 (N[1339) 2001e2005 (N[2254) 2006e2010 (N[1274)
Difference
between
the three
groups,
p Value
Mean (SD) Median (5e95) Mean (SD) Median (5e95) Mean (SD) Median (5e95) Mean (SD) Median (5e95)
Been treated for
Varicocele 0.4 0.0 0.7 0.4 0.004***
Testicular torsion 0.8 1.0 0.6 0.9 0.4***
Testicular cancer 0.02 0.0 0.0 0.1 0.2***
Cryptorchidism** 6.1 3.9 8.0 5.0 <0.0005***
Hypospadias 0.1 0.0 0.0 0.3 0.004***
Phimosis 3.9 5.4 2.7 4.6 <0.0005***
Inguinal hernia 3.4 3.8 4.8 0.5 <0.0005***
Has
Had cryptorchidismyy 8.3 4.4 11.2 6.9 <0.0005***
Experienced fertility
problemszz
0.6 1.7 0.2 0.2 <0.0005***
Caused a
pregnancyxx
6.4 7.4 5.6 6.8 0.08***
Subgroup 80.6 84.6 77.8 81.2 <0.0005***
*Calculated as difference between day of attendance in study and self-reported day of birth.
yMean of left and right testes size assessed by palpation. Information of testis size was missing for 3, 9 and 3 men from the 1st, 2nd and 3rd investigation period, respectively.
zSum of intake of beer, wine and strong alcohol recent week prior to participation in study.
xTaken any medication recent 3 months prior to participation in study.
{Chlamydia or gonorrhoea.
**Hormonal, surgical or combination.
yyNot born with both testicles in scrotum (irrespective of spontaneous descend, treatment or still cryptorchid).
zzEver had regular intercourse without use of contraception for at least 1 year, without partner became pregnant.
xxEver caused a pregnancy.
{{KruskaleWallis test.
***
c
2
test.
Subgroup: Men without adverse conditions ‘Been diagnosed as having.’, ‘Been treated for.’ or ‘Has.’. Those that have caused a pregnancy are also included, irrespective of any adverse
condition previously. See text for further explanation.
p Value: For comparison of results between the three study periods.
4 Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990
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Semen samples
Semen samples were produced by masturbation. The
actual abstinence period was calculated from the self-
reported time of previous ejaculation and the time of
delivery of semen sample recorded by a technician. The
semen samples were produced in the privacy of a room
near the laboratory and kept at 378C.
Semen analysis was performed according to the WHO
guidelines.
36
In brief, semen volume was estimated by
weighing the collection tube with the semen sample and
subtracting the predetermined weight of the empty tube,
assuming that 1 ml semen ¼1 g. For sperm motility
assessment, 10 ml of well-mixed semen was placed on
a clean glass slide kept at 378C and covered with
a22322 mm coverslip. The preparation was placed on
the heated stage of a microscope at 378C and immediately
examined at 3400 magnification. The sperm were clas-
sified as progressively motile, locally motile or immotile.
For the assessment of the sperm concentration, the
samp
les were diluted in a solution of 0.6 mol/l NaHCO
3
and 0.4% (v/v) formaldehyde in distilled water. The
sperm concentration was subsequently assessed using
aBu
¨
rker-Tu
¨
rk haemocytometer (Paul Marienfeld GmbH
& Co. KG, Lauda-Ko
¨
nigshofen, Germany). Only sperm
with tails were counted.
Smears were prepared for morphological evaluation,
Papanicolaou stained and finally assessed according to
‘strict criteria’.
37
The laboratory participates in an external quality
control programme for sperm concentration assessment
as previously described.
15 38e40
The results did not show
any temporal trend in assessment level in the Copen-
hagen laboratory. Therefore, no adjustments according
to quality control results were needed. For the first 290
men included in our study, the weight of the empty
semen collection tubes was on average overestimated by
0.5 g, giving an underestimation of the semen weight by
0.5 g. The data on semen volum e among these men were
therefore corrected as follows: Corrected semen volume
¼ Observed semen volume +0.5 ml.
Physical examination
A physical examination of each participant was
per
formed on the day of delivery of his semen sample.
Tanner stage of pubic hair was recorded, and testicular
size was assessed, all examiners using the same type of
wooden orchidometer.
Comparison population: fertile men (partners of pregnant
women), examined in 1996e1998
From October 1996 to January 1998, our group also
ex
amined the semen quality of 349 fertile men (partners
of pregnant women); the results were published previ-
ously.
12
Pregnant women were approached during
routine visits at the antenatal care unit, and their partners
were invited to participate in the semen quality study. The
eligibility criteria were age 20e45 years at the time of
invitation, both the man and his mother being born and
raised in Denmark and conception of the current preg-
nancy by normal sexual relations (not as a result of
treatment for subfertility or infertility). Participation of
these men was similar to that of the men from the general
population: they answered a questionnaire, delivered
a semen sample and had a physical examination
performed. Both physical examination and semen anal-
ysis were performed in the same manner and in the same
laboratory as for men from the general population.
A description of the fertile men based on question-
naire information and on the results of the physical
examination has previously been published
12
and is
shown in table 2, which also includes additional infor-
mation to allow for a better comparison to the men from
the general population.
Comparison population: male partners from infertile
couples, examined in 1939e1943
Dr Richard Hammen published a doctoral thesis with
gro
und-breaking data on male infertility in 1944.
13
From
the Copenhagen area in Denmark, he investigated 925
men in ‘childless marriages’, defined as couples where at
least 1 year of regular coitus, without use of contracep-
tives, had not led to a successful pregnancy.
Hammen’s data origi nated from two cohorts (‘mate-
rial
I’ and ‘material II’) that he examined in 1939 e 1943.
Material I comprised 291 male partners attending the
Gynaecological Departmen t and Dispensary of the
Kommune Hospital, Copenhagen. Material II consisted
of 634 men who delivered basic information and semen
sample to the General Laboratory of National Health
Insurance Physicians, Copenhagen. Hammen stated that
information regarding duration of childlessness was
somewhat less reliable in this group than in material I
but concluded that only a few per cent of these men had
a duration of childlessness <1 year.
In his thesis, Hammen provided patient histories of
the study populations. The durations of childlessness in
the cohorts were 1e2 years (12.7% ), 2e3 years (22.7%)
and more than 3 years (64.4%). Secondary sterility was
ascertained in 26.8%, as 12.0% had children or abor-
tuses with other women and 14.8% with their current
partners.
The age distribution of the whole Hammen cohort
(materials I and II) was described as 2.7% <25 years,
72.6% 25e35 years, 22.3% 35 e 45 years and 2.4%
>45 years. Therefore, the median age seems to be
somewhere in early 30s, similar to that of the fertile men
we investigated in 1996e1998.
Some of the information obtained by Hammen was
similar to that obtained in our studies of men from the
general population and partners of pregnant women;
this is summarised in
table 2.
Information on medical
history was obtained from all men in material I, but only
from 548 men from material II. Thus, the figures
presented in table 2 are calculated based on information
from 839 men.
Hammen did not report height or weight, but noted
‘mode
rate or marked obesity’ in 6.6% of the men.
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Palpable changes in the epididymis were detected in
11.8%, abnormalities of the testis in 25.2%, cryptorchidism
of one or both testes in 5.2% and varicocele in 11.9%.
Previous venereal diseases were reported by 24.1% of
men in material I and 16.9% in material II. Other local
lesions involving the testes were reported (eg, hernia,
cryptorchidism) in 12.1% in material I and 4.0% in
material II. Hammen attributed the difference between
the two otherwise similar groups to erroneous informa-
tion given by the questioned patients in material II, as
they were not interviewed by Hammen directly. Previous
serious diseases (eg, pulmonary tuberculosis,
Table 2 Physical appearance and self-reported information of men from the Copenhagen area, Denmark
Study population
General population
1996e2010, total
(N[4867)
Fertile 1996e1998
(N[349)
Infertile couples
1939e1943
(N[839)
Mean (SD) Median (5e95) Mean (SD) Median (5e95)
Physical appearance
Age (years)* 19.4 (1.2) 19.0 (18.4e21.7) 31.5 (4.3) 30.8 (25.4e40.2) 73% 25e35 years
Height (m) 1.81 (0.06) 1.81 (1.71e1.92) 1.83 (6.2) 1.84 (1.73e1.94) e
Weight (kg) 75.1 (11.5) 73.5 (59.4e96.4) 83.0 (11.2) 82.0 (67.6e102.2) e
BMI (kg/m
2
) 22.9 (3.1) 22.4 (18.7e28.8) 24.6 (2.9) 24.3 (20.6e29.5) 6.6% ‘moderate
obese’
Testis size (ml)y 20 (5) 20 (13e28) 23 (4) 24 (15e30) e
Testis size (ml), US 15 (4) 14 (9e22) ee e
Lifestyle
Cigarettes daily, all men 4.1 (6.7) 0.0 (0.0e20.0) 4.5 (8.3) 0.0 (0.0e20) e
Cigarettes daily, smokers only 9.9 (7.1) 10.0 (1.0e20.0) 14.0 (8.9) 15.0 (0.5e30) e
Alcohol consumption (units)z 14 (14) 11 (0e40) 10 (9) 8 (0e30) e
Ejaculation abstinence (h) 81 (117) 63 (37e155) 81 (65) 64 (20e182) e
%% %
Taken medicationx 12.5 20.1 e
Smoker 41.7 32.5 e
Previous smoker 3.1 ee
Never-smoker 55.2 ee
Mother smoked in pregnancy 38.0 38.1 e
Been diagnosed as having
Varicocele 0.6 2.9 11.9
Epididymitis 0.3 2.6 1.9
Sexual transmitted disease{ 4.3 18.6 19.4
Cystitis 2.4 8.0 e
Diabetes 0.02 0.3 e
Thyroid disease 0.04 0.0 e
Been treated for
Varicocele 0.4 0.9 0.4
Testicular torsion 0.8 1.1
Testicular cancer 0.02 0.3 0.01
Cryptorchidism** 6.1 4.3 2.1
Hypospadias 0.1 0.0 0.0
Phimosis 3.9 ee
Inguinal hernia 3.4 6.0 1.5
Has
Had cryptorchidismyy 8.3 e >5.2
Experienced fertility problemszz 0.6 12.3 100.0
Caused a pregnancyxx 6.4 100.0 26.8
Infertile couples: 925 men delivered semen samples, however, patient history was only obtained on 839.
*Calculated as difference between day of attendance in study and self-reported day of birth.
yMean of left and right testes size assessed by palpation. Information of testis size was missing for 3, 9 and 3 men from the 1st, 2nd and 3rd
investigation period, respectively.
zSum of intake of beer, wine and strong alcohol recent week prior to participation in study.
xTaken any medication recent 3 months prior to participation in study.
{Chlamydia or gonorrhoea.
**Hormonal, surgical or combination.
yyNot born with both testicles in scrotum (irrespective of spontaneous descend, treatment or still cryptorchid). For the Hammen cohort similar
information was not obtained, but 5.2% of men were detected as having cryptorchidism when examined.
zzEver had regular intercourse without use of contraception for at least 1 year, without partner became pregnant.
xxEver caused a pregnancy.
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pneumonia and peritonitis) were recorded in 34.7% of
the cases. Present chronic conditions (diseases of the
stomach and intestines, especially peptic ulcer and
gastritis, neurasthenia, chronic otitis media, chronic
familial anaemia, osteomyelitis, bronchitis, diabetes
mellitus, epilepsy and heart disease) were recorded in
12.7% of the patients.
All men provided a semen sample. They had been
instructed to abstain from ejaculation for at least 3 days
prior to delivery of the semen sample, which was
produced throu gh interrupted coitus at home or by
masturbation in a private room in the hospital. Exact
information was requested about the period of ejacula-
tion abstinence.
The ejaculates were examined as soon as the specimen
was received. Semen was poured from the collection
beaker into a graduated container to assess volume. For
assessment of sperm concentration, the following
procedure was employed: ‘After thorough mixing of the
sperm in a shaking-machine or by hand, 0.1 cc. of sperm
is added to 1.9 cc. of the diluent (consisting of 190 cc. of
physiological salt solu tion, 7 cc. of a 1% methylene blue
solution and 3 cc. of absolute alcohol). The mixing takes
place in a dwarf-tube, containing a glass bead, which is
shaken for 5 m in in a shaking-ma chine or by hand. The
count is carried out with employment of a Bu
¨
rker-Tu
¨
rk
counting-chamber. All sperm heads are counted, also
loose heads. Loose tails are not counted’.
Statistical analysis in the present study
Means, medians, SDs, 5e95
percentiles and frequencies
were used for basic descriptions. The study subjects were
divided into three groups, depending on the investiga-
tion periods: 1996e2000, 2001e 2005 and 2006e2010.
Between-group differences for continuous variables were
tested by the non-parametric KruskaleWallis test.
Between-group differences for categorical variables were
tested with Pearson’s
c
2
test.
The main outcome variables were semen volume,
sperm concentration, total sperm count, percentage of
motile spermatozoa and percentage morphologically
normal spermatozoa. Temporal trend between investi-
gation periods were tested by multiple linear regressions
adjusted for confounders. Semen volume, sperm
concentration and total sperm count were best normal-
ised by a cubic root transformation before analysis to
correct for skewed distribution of residuals. The
percentages of motile spermatozoa were logit-trans-
formed. Percentages of morphologically normal sper-
matozoa were close to normally distributed and entered
the model untransformed. Ejaculation abstinence up to
96 h had an increasing effect on semen volume, sperm
concentration and total sperm count (all p values<0.05)
and was entered as a covariate in the regression analyses
of these variables, whereas it had no effec t on
morphology or motility. Increasing age had a non-
significant increasing effect on sperm concentration
(p¼0.8), but a significant increasing effect on semen
volume (p<0.0005), and was also entered as covariate.
Season of year was evaluated as a possible confou nder
for all the semen variables, and duration from ejacula-
tion to assessment was additionally evaluated as
a confounder for sperm motility. Both were non-signifi-
cant and therefore not included in the final models.
Differences in semen quality variables between men
from the general population and partners of pregnant
women were also tested by linear regressions corrected
for the same covariates as stated above. Differen ces in
distribution of sperm concentrations and total sperm
counts between men from the general pop-
ulation examined in 1996e2010 and male partners from
infertile couples examined in 1939e1943 were tested by
c
2
test.
A p value of <0.05 was considered statistically signifi-
cant. Analyses were performed using PASW GradPack
V.18.0 (SPSS Inc.).
RESULTS
Figure 1 shows sperm concentrations, total sperm counts
and percentages of morphologically normal sperma-
tozoa for each year of examination. Grouping results of
the 15 years into three 5-year periods showed a temporal
increase in the sperm counts (table 3). Men examined in
2006e2010 had higher median sperm concentration,
total sperm count and total number of morphologically
normal sperm count than men examined in the first 5-
year period. Similarly, men examined in 2001e2005
appeared to have higher counts than the previously
examined. Estimating the average linear increase over
the period confirmed the slightly increasing temporal
trends (p¼0.02, p<0.0005 and p¼0.013, respectively).
The median values indicated an increase in semen
volume, which was confirmed both when the three 5-year
periods were compared and when estimating the annual
linear increase (p<0.0005). The percentages of motile
and morphologically normal spermatozoa showed no
change over time.
As expected, some of the men had previously experi-
enced andrological problems, including cryptorchidism,
hypospadias, sexually transmitted diseases and/or other
signs or symptoms relating to the reproductive system
(
table 1).
As men with such diseases could be more
motivated to participate in the study, we performed
a subanalysis on the subgroup of 3921 men (80.6%) who
were without previous andrological abnormalities. The
main conclusion that impaired semen quality was
frequent remained robust. The results described here
are based on the entire group, whereas the results from
the subgroup are shown in appendix 1.
Comparison population: fertile men, examined in
1996e1998
Table 4 summarises the semen results of the 349 fertile
men examined previously
12
and the men from the general
population. The semen variables differed between these
groups, with highest semen volume, sperm concentration,
total sperm count, total number of morphologically
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 7
Human semen quality in the new millennium
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normal spermatozoa and percentage of normal sperma-
tozoa in the fertile men (all p values <0.0005). Motility
variables were statistically lower for the fertile men.
Approximately 42% of the men from the general pop u-
lation had sperm concentrations below 40 million/ml
and 66% had <9% normal forms (figure 2). For 15% and
35% of men, the sperm concentration was below
15 million/ml and the percentage of normal spermatozoa
below 5%. For the fertile men, only 8% had a sperm
concentration below 15 mill/ml and 18% had <5%
normal forms. Only 23% of men from the general popu-
lation had the optim al sperm concentrations of more
than 40 million/ml and more than 9% normal forms, in
comparison to 42% of the fertile men.
Comparison population: male partners from infertile
couples, examined in 1939e1943
Figure 3 shows the distributions of sperm concentrations
and total sperm counts for our study group compared
with those of men from infertile couples in Denmark in
1940e1943 (Hammen cohort). As shown, our study
group had lower sperm counts than the historical cohort
(p<0.0005, for all comparisons).
DISCUSSION
In this large, prospective and well-controlled stud y of
semen quality of annual cohorts of young men from the
general population, statistically significant increases in
sperm concentration and total sperm counts over the
past 15 years were dete cted. However, it is of concern
that these men from the general population in the new
millennium had significantly lower sperm concentra-
tions and total sperm counts than recently examined
fertile men and men of a historical cohort of male
partners of infertile couples. Both sperm concentration
and sperm morphology measures according to strict
criteria are known to be informative semen measure-
ments for discriminating between fertile and infertile
men.
41
Therefore, there is reason to be concerned about
future fertility of young Danish men. Smaller cro ss-
sectional studies of men from the general populations in
other European countries have shown similar high
frequencies of men with poor semen quality.
38e40 42 43
Thus, poor semen quality seems to be a widespread
phenomenon. This interpretation is in line with the high
and increasing need of fertility treatment in Denmark.
44
We have considered whether immaturity of the men
(with a median age of 19 years) could account for the
findings. However, a 4-year longitudinal follow-up with
quarterly assessment of semen quality in a subgroup of
more than 150 of the men showed no significant change
over time in sperm concentration, total sperm count and
sperm morphology, suggesting that immaturity does not
explain our results.
45
It is also possible that our results
could be skewed by selection biases. However, during the
early stage of our project, we carried out a study on
blood samples from the majority of those men who did
not volunteer to provide semen samples (N¼195,
participation rate 79%) and showed that their repro-
ductive hormone levels including the spermatogenesis
markers follicle-stimulating hormone and inhibin-b were
very similar to those of the participants.
14
This suggests
that our results are not biased by selection. Furthermore,
our results hold true in the subgroup of men without
andrological events in their history as presented in
appendix 1. It is not likely that the detected temporal
trend in sperm count is due to intraobserver or inter-
observer variations. Our laboratory technicians partici-
pated in a quality control study of assessment of sperm
concentration, which did not indicate temporal changes
Figure 1 Semen parameters of Danish men from the general
population. Red bars show 25the75th percentiles with median
line. Whiskers show 5the95th percentiles. The sperm
concentration (A) and total sperm count (B) increased slightly
by year of examination. Percentage of morphologically normal
spermatozoa did not show any temporal trend (C).
8 Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990
Human semen quality in the new millennium
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in assessment levels. However, a longer observation
period is needed to corroborate or refute such a positive
tendency. Five observers did 97.6% of all morphology
assessments, among which a single observer assessed
91% of samples in the first 5-year period, 14% in the
second period and none in the last. One observer
assessed 14% of samples from the second period, but
none from the first or last period. This obser ver tended
to obtain 1% lower values than other observers (detailed
data not shown), which partly explains the lower number
of morphologically normal spermatozoa in the second 5-
year period. Assessment of semen volume was also
controlled and corrected when needed. Effects of
potential confounders of semen variables were investi-
gated and accounted for in the stat istical analyses.
Increasing duration of abstinence up to approximately
96 h had an increasing influence on semen volume,
sperm concentration and total sperm count, but no
effect on motility or morphology, which is in agreement
with our initial findings and with the results of oth er
semen quality studies of men from Europe.
38e40 42
From
pilot studies in the middle of 1990s, we know that
interobserver variation for motility assessment is of
significant importance
46
and difficult to eliminate. Our
results on numbers of motile sperms should therefore be
taken with som e caution.
The definition of normal semen quality has varied over
time
. Seventy years ago, the Danish standard for normal
sperm concentration set 60 million/ml as a lower cut-off
level.
13
However, the most recent WHO guid elines
adhere to common clinical practice, where the ‘normal’
reference range is defined as the one that covers 95% of
a population. The most recent WHO guidelines have
reduced the reference limits for sperm concentration
Table 3 Semen quality of 4867 young men from the general population in the Copenhagen area in Denmark
Mean (SD) Median (5e95)
p Values comparing
All periods
2001e2005 vs
2006e2010
1996e2000 vs
2006e2010
Semen volume (ml)
Investigation period 1996e 2010 3.4 (2.0) 3.2 (1.3e6.0)
Investigation period 1996e 2000 3.3 (1.5) 3.1 (1.2e5.8)
Investigation period 2001e 2005 3.3 (1.4) 3.2 (1.4e5.9)
Investigation period 2006e 2010 3.6 (3.1) 3.3 (1.3e6.3) 0.004 0.011 0.001
Sperm concentration (million/ml)
Investigation period 1996e 2010 60 (57) 45 (3 e 163)
Investigation period 1996e 2000 58 (55) 43 (3e167)
Investigation period 2001e 2005 60 (58) 45 (3e156)
Investigation period 2006e 2010 62 (55) 48 (3e169) 0.065 0.12 0.020
Total sperm count (million)
Investigation period 1996e 2010 193 (232) 143 (9e 529)
Investigation period 1996e 2000 185 (184) 132 (6e531)
Investigation period 2001e 2005 191 (241) 146 (8e508)
Investigation period 2006e 2010 206 (258) 151 (13e559) 0.002 0.015 0.001
Normal morphology (%)
Investigation period 1996e 2010 7.1 (4.9) 6.5 (0.5e16.0)
Investigation period 1996e 2000 7.3 (5.1) 7.0 (1.0e17.0)
Investigation period 2001e 2005 6.9 (4.8) 6.0 (0.5e15.5)
Investigation period 2006e 2010 7.5 (4.9) 7.0 (0.5e
16.0) 0.016 0.023 0.97
Total
normal spermatozoa (million)
Investigation period 1996e 2010 16.3 (23.9) 8.4 (0.0e57.4)
Investigation period 1996e 2000 16.5 (24.5) 7.9 (0.0 e 60.9)
Investigation period 2001e 2005 15.5 (22.6) 8.0 (0.0 e 53.8)
Investigation period 2006e 2010 17.9 (25.3) 9.8 (0.1 e 59.3) 0.040 0.012 0.076
Progressively motile (%)
Investigation period 1996e 2010 56 (17) 59 (23e 77)
Investigation period 1996e 2000 54 (17) 57 (22e75)
Investigation period 2001e 2005 57 (17) 60 (22e77)
Investigation period 2006e 2010 57 (16) 59 (25e79) <0.0005 0.30 0.005
Motile (%)
Investigation period 1996e 2010 65 (15) 68 (35e 83)
Investigation period 1996e 2000 65 (15) 68 (38e82)
Investigation period 2001e 2005 64 (15) 67 (35e82)
Investigation period 2006e 2010 65 (16) 68 (33e85) 0.17 0.09 0.71
5e95: 5e95 percentiles.
p Values: Obtained from regression analysis taking confounders into consideration.
Jørgensen N, Joensen UN, Jensen TK, et
al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 9
Human semen quality in the new millennium
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from 20 to 15 million/ml.
36
Reference limits based on
95% of the population may be rel evant in relation to
certain clinical tests (eg, levels of sodium or potassium in
serum) but are unsuitable for public health issues in
which secular changes may affect the whole population
(eg, obesity).
47
For trend analyses, our data on semen
quality of men examined during the past 15 years should
therefore rather be compared with data from the previous
generations of men. Unfor tunately, historic al data on
semen quality of men from the general population do not
exist. Other unique Danish semen data obtained by the
pioneer of modern Danish andrology, Dr Richard
Hammen, who studied semen quality of men 70 years
ago, exists.
13
His method for counting sperm concentra-
tion by the use of the Bu
¨
rker-Tu
¨
rk haemocytometer was
very similar to that used in our present investigations and
in accordance with the current recommen dations by the
WHO,
36
allowing for meaningful comparisons with our
new data. Interestingly, sperm num bers among men in
the Hammen study from the 1940s were significantly
higher than those in the present study, despite the fact
that the earlier sample was recruited among male part-
ners in infertile couples. This actually corroborates that
semen quality might have decreased temporarily as
suggested by the meta-analysis by Car lsen et al.
1
Whereas the historical data point to a temporal
decrease in sperm concentration and total sperm counts,
there is no such data to support a similar trend in the
percentages of normal spermatozoa in each ejaculate. A
trend may be difficult to detect because different criteria
for normality have been applied during the years. In our
study, we did not find any trend in sperm morphology
despite a slight increase in sperm numbers. However, it is
noteworthy that the median percentage of spermatozoa
with normal morphology was as low as 6.5%. In contrast
to our study, a decrease in the percentage of normal
spermatozoa was recently described in a Finnish study,
which also reported decreasing trends for sperm
concentration and total sperm counts.
40
Although only one spermatozoon is needed to fertilise
an egg, several studies have shown that the fertilising
ability diminishes if the sperm concentration is belo w
40e50 million/ml or if the percentage of normal sper-
matozoa is below 9%.
48e51
Approximately 42% of the
men from the general population had sperm concen-
trations below 40 million/ml and 66% <9% normal
forms. More sever e fertility problems may be present
when sperm concentration is below 15 million/ml and
the percentage of normal spermatozoa is <5%,
41 51
which was the cas e for 15% and 35% of the men from
the general Danish population, respectively. It is note-
worthy that only 8% and 18% of a group of fertile men in
a previous study of partners of pregnant women were
below these ‘cut-off’ levels. Spe rm concentration, total
sperm count and percentage of normal spermatozoa
were significantly lower in men from the general popu-
lation in comparison to fertile men. Only 23% of men
from the general population had the optimal sperm
concentrations of more than 40 million/ml and more
than 9% normal forms in comparison to 42% of the
fertile men.
Both clinical practice and animal studies suggest an
imp
ortant role of sperm morphology for conce ption
rates.
41 51
Human in vitro fertilisation studies also
suggest an important role of sperm morphology for
fertilisation rates, which become significantly lower if the
percentage of normal spermatozoa is below 5%. In men,
the number of morphologically normal spermatozoa is
usually reported to be below 10% and in animals above
50%. For example, breeding bulls and boars mos t often
have <10% abnormal spermatozoa,
52
and abnormalities
are often more subtle than the severe abnormalities
frequently seen in human samples. Even with relatively
low numbers of normal spermatozoa, humans may still
be able to reproduce. In contrast to wild animal species,
where survival of the species may depend on a very high
conception rate at each coitus, humans in monogamous
relationships are not dependent on immediate
Table 4 Semen quality of partners of pregnant women (fertile men) and young men from the general population from the
Copenhagen area in Denmark
Partners pregnant
women (N[349)
General population
Total group (N[4867) Subgroup (N[3921)
Mean (SD) Median (5e95) Mean (SD) Median (5e95) Mean (SD) Median (5e95)
Semen volume (ml) 3.8 (1.7) 3.6 (1.1e 6.7) 3.4 (2.0) 3.2 (1.3e6.0) 3.4 (2.1) 3.2 (1.3e5.9)
Sperm concentration
(million/ml)
77 (66) 61 (10e207) 60 (57) 45 (3e163) 61 (57) 47 (4e166)
Total sperm count (million) 276 (240) 215 (32e795) 193 (232) 143 (9e529) 197 (231) 146 (10e531)
Normal morphology (%) 9.3 (5.0) 8.5 (2.0e 18.5) 7.1 (4.9) 6.5 (0.5e16.0) 7.2 (4.9) 6.5 (0.5e16)
Total normal spermatozoa
(million)
30 (37) 18 (1 e 111) 16 (24) 8 (0e57) 17 (24) 9 (0.1e59)
Progressive motile (%) 51 (15) 52 (25e72) 57 (16) 60 (24e77) 57 (16) 60 (24e77)
Motile (%) 60 (12) 61 (40e79) 65 (15) 68 (35e 83) 65 (15) 68 (37e83)
5e95: 5e95 percentile.
Subgroup: Men without adverse conditions ‘Been diagnosed as having.’, ‘Been treated for.’ or ‘Has.’ as described in table 1. Those that
have caused a pregnancy are also included, irrespective of any adverse condition previously. See table 1 and text for further explanation.
10 Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990
Human semen quality in the new millennium
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reproductive success to the same degree. In fact, the
current definition of couple infertility in most national
health systems is ‘more than 1 year of regular, unpro-
tected sexual relationship without pregnancy’.
53
In other
words, absence of pregnancy in spite of regular coitus
during up to 12 ovulation periods can be considered
‘normal’ from a clinical point of view. However, fecun-
dity m ay still be reduced compared to couples where
conception occurs immediately after unpr otected inter-
course during the first cycle.
In conclusion, our large prospective study of men of
the
general population supports previous suggestions of
a temporal decrease in semen quality, but it also indi-
cated a recent smal l increase in sperm concentration
and total sperm count. Follow-up studies are needed to
detect if the upward trend is a real biological phenom-
enon or merely random variation. It is noteworthy that
only one in four men had optimal semen quality from
Figure 2 Distributions of sperm counts and morphologically
normal spermatozoa in Danish men from the general
population and fertile Danish men (partners of pregnant
women). All men had durations of ejaculation abstinence
above 48 h. Sperm concentration (A) total sperm counts (B)
and percentages of morphologically normal spermatozoa (C)
were lower in men from the general population.
Figure 3 Distributions of sperm counts in Danish men from
the general population, examined from 1996 to 2010 and
Danish men examined in an infertility clinic in the 1940s. All
men had durations of ejaculation abstinence above 48 h.
Sperm concentration (A) and total sperm counts (B).
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 11
Human semen quality in the new millennium
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a fecundity perspective. Approximately 25% had
a reduced quality compatible with prolonged waiting
time to pregnancy, and another 15% had so severely
impaired quality that they have a high risk of the need
for fertility treatment to become biological fathers.
Author affiliations
1
University Department of Growth and Reproduction, Rigshospitalet,
University of Copenhagen, Copenhagen, Denmark
2
The Fertility Clinic Rigshospitalet, Copenhagen, Denmark
3
Institute of Public Health, Department of Biostatistics, University of
Copenhagen, Copenhagen, Denmark
4
Department of Physiology, University of Turku, Turku, Finland
5
Department of Paediatrics, University of Turku, Turku, Finland
Contributors Substantial contributions to conception and design: NJ, TKJ,
A-MA, JT and NES. Acquisition of data: NJ, UNJ, TKJ, MBJ, IAO, AJ and EC.
Analysis of data: NJ, UNJ, KA, JHP and NES. Interpretation of data: all authors.
Drafting the article: NJ, UNJ and NES. Revising the article critically for
important intellectual content: all authors. Final approval of the version to be
published: all authors.
Funding This study has been supported economically by several grants: the
European Union (contract numbers BMH4-CT96-0314,
QLK4-CT-1999e01422, QLK4-CTd2002e00603 and most recently FP7⁄
2007e2013, DEER Grant agreement no. 212844), the Danish Research
Council (grants numbers 9700833 2107-05-0006), the Danish Agency for
Science, Technology and Innovation (Grant number 271070678),
Rigshospitalet (Grant number 961506336), the University of Copenhagen
(Grant number 211-0357/07-3012), the Danish Ministry of Health and the
Danish Environmental Protection Agency, A.P. Møller and wife Chastine
McKinney Møllers foundation and Svend Andersens Foundation. The funding
organisations played no role in the design and conduct of the study; in
collection, management, analysis and interpretation of the data; or in the
presentation, review or approval of the manuscript.
Competing interests None.
Ethics approval The local Science Ethical Committee had approved the study
(June 1996, the Science Ethical Committee for the Copenhagen and
Frederiksberg municipalities, reference number KF01-117/96, and most
recently June 2011, the Capital Region of Denmark, reference number
H-KF-289428), and all participants had given their informed consent.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement There are no additional data available.
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APPENDIX 1
Semen results for men from the general population have been
summarised in
figure 1 of
the main text. Online table A1 summarises
the semen results for the subgroup of men without previous andro-
logical abnormalities as well as p values for differences between the
three 5-year periods (similar to table 2, which described the entire
study population in the main text). Men examined in 2006e2010 had
higher median sperm concentration, total sperm count and total
number of morphologically normal spermatozoa than men examined in
the first 5-year period. In analyses using year of examination as
a continuous variable, the significant trends were also confirmed for the
subgroup: p¼0.02, p¼0.001 and p¼0.004.
Table 3 in the main text summarised the semen results of the 349
fertile men examined previously.
12
The semen variables differed
between these men and men from the general population, with highest
semen volume, sperm concentration, total sperm count, total number of
morphologically normal spermatozoa and percentage of normal sper-
matozoa in the fertile men. These differences were all highly significant
at p<0.0005, irrespective of the comparisons being made between the
fertile men and the entire study group of men from the general popula-
tion; the subgroup of men from the general population without any
andrological event in their history examined in 1996e2010 or the smaller
subgroup examined in 2006e2010. The differences are shown in online
figure A1.1 and A1.2, which show the distributions of sperm concentra-
tions, total sperm count and number of morphologically normal sper-
matozoa for men from the general population (red bars), and partners of
pregnant women (green bars). For these variables, we show data based
on the entire study population, data based on only those with an ejac-
ulation abstinence of at least 48 h, data based on only those having an
ejaculation abstinence period of at least 48 h and without any andro-
logical event in their history (subgroup) and finally data based on the
same subgroup of men from the general population examined in the
period 2006e2010. The tendency that men from the general population
have lower semen volume, sperm concentration, total sperm count, total
number of morphologically normal spermatozoa and percentage of
normal spermatozoa than partners of pregnant women is seen irre-
spective of which of the four groupings are evaluated.
Online figure A2 shows the distributions of sperm concentrations and
total
sperm counts for the men from the general population (grouped as
in online figure A1) compared with men from infertile couples in
Denmark, 1940e1943.
13
Here, too, it can be seen that the recent
general population has lower sperm counts than the historical cohort
(p<0.0005, for all comparisons).
PAGE fraction trail=12.75
Jørgensen N, Joensen UN, Jensen TK, et al. BMJ Open 2012;2:e000990. doi:10.1136/bmjopen-2012-000990 13
Human semen quality in the new millennium
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doi: 10.1136/bmjopen-2012-000990
2012 2: BMJ Open
Niels Jørgensen, Ulla Nordström Joensen, Tina Kold Jensen, et al.
population-based study of 4867 men
millennium: a prospective cross-sectional
Human semen quality in the new
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