East–West gradient in semen quality in the Nordic–Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland

Niels Jørgensen1,7, Elisabeth Carlsen1, Ingrid Nermoen2, Margus Punab3, Jyrki Suominen4, Anne-Grethe Andersen1, Anna-Maria Andersson1, Trine B. Haugen2, Antero Horte4, Tina Kold Jensen1,5, Øystein Magnus2, Jørgen Holm Petersen1,6, Matti Vierula4, Jorma Toppari4 and Niels E. Skakkebæk1

1 Department of Growth and Reproduction, Rigshospitalet, The Juliane Marie Centre, Copenhagen, Denmark, 2 Department of Obstetrics and Gynecology, Rikshospitalet, Oslo, Norway, 3 Clinics of Surgery, Tartu University Clinicum, Tartu, Estonia, 4 Institute of Biomedicine, Departments of Anatomy, Physiology and Paediatrics, University of Turku, Turku, Finland, 5 Institute of Public Health, Department of Environmental Medicine, University of Southern Denmark, Odense, Denmark and 6 Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Denmark and Norway have a three-fold higher incidence of testicular cancer than Estonia and Finland. Groups of young men from Denmark, Norway, Finland and Estonia were investigated to elucidate whether semen parameters and other related parameters follow a gradient between these countries, as does the gradient in incidence of testicular cancer. METHODS: In total, 968 young men from the general population in these four countries were investigated according to the same protocol. Possible confounders were evaluated, and included in the statistical analysis when appropriate. Inter-laboratory differences in assessment of sperm concentrations were controlled by an external quality control programme and morphology assessment was centralized to one person. RESULTS: The Finnish and Estonian men had an adjusted median sperm concentration of 54 and 57x106/ml, respectively and the Norwegian and Danish men 41x106/ml. The corresponding total sperm counts were 185, 174, 133 and 144x106. The frequency of normal sperm in men from Finland was 8.9%, Estonia 9.2%, Norway 6.9% and Denmark 6.4%. Within all four groups of men, a relationship between increasing levels of inhibin-B and increasing sperm counts was observed. However, inhibin-B levels were not predictive of sperm count differences between countries. CONCLUSIONS: It is believed that the men examined were representative of the normal population of young men in all four countries as they were recruited from groups attending a compulsory medical examination, and not selected for known fertility or semen quality. Moreover, the majority of participants had no prior knowledge of their fertility potential. It appears that an east–west gradient exists in the Nordic–Baltic area with regard to semen parameters, this being in parallel with the incidences of testicular cancer. Further investigations are required to determine whether these findings are due to genetic differences, to different environments, or perhaps to a combination of both factors.

Key words: reference group/regional differences/semen quality/sex hormones


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Male reproductive health has been in focus during recent years, with several laboratories having reported deterioration in semen quality (Carlsen et al., 1992Go; Auger et al., 1995Go; Irvine et al., 1996Go; Van Waeleghem et al., 1996Go). Although endocrine disrupters have been suggested as one of the possible causes (Toppari et al., 1996Go; Skakkebaek et al., 2001Go), the topic remains controversial and the adverse trend is not apparent everywhere (Suominen and Vierula, 1993Go; Bujan et al., 1996Go; Fisch et al., 1996Go; Paulsen et al., 1996Go; Vierula et al., 1996Go). Testicular cancer represents another aspect of male reproductive health, and in the Nordic–Baltic area there is a remarkable east–west gradient in the incidence of this disease. Finland and Estonia have a very low testicular cancer incidence which is less than one-third of that in Denmark and Norway (Adami et al., 1994Go). Recently, it was shown that young men from the general Danish population had a surprisingly low sperm concentration (median 45x106/ml) (Andersen et al., 2000Go). Based on retrospective and not directly comparable data, Finnish men have previously been reported to have a high and unchanged mean sperm concentration (94–114x 106/ml) (Suominen and Vierula, 1993Go; Vierula et al., 1996Go).

According to a recently proposed hypothesis, testicular cancer does not occur at random but in men with testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001Go). Depending on severity, TDS may include one or more disorders, such as impaired spermatogenesis, testicular cancer, hypospadias and undescended testes. This hypothesis implies that populations with high risk of testicular cancer also have an increased frequency of men with poor semen quality, and vice versa. This hypothesis was corroborated by one study (Andersen et al., 2000Go) in which low sperm concentrations were found among young Danish men who had the highest rate of testicular cancer. Thus in the present study, groups of young men from Denmark, Norway, Finland and Estonia were investigated to determine whether, in these countries, semen parameters and sex hormone levels follow a similar gradient to the incidence of testicular cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population
In Finland, Estonia, Norway and Denmark all young men, except those suffering from chronic severe diseases (<15%), are required to attend a compulsory medical examination before they may be considered for military service. Therefore, such an attending group of men may be considered representative of the general population of young men in their country.

The opportunity was acquired to collaborate with the military health authorities in the cities of Turku in Finland, Tartu in Estonia, Oslo in Norway and Copenhagen in Denmark. Young men from these cities were enrolled into the present study when they attended the compulsory medical examination, irrespective of whether they were declared fit for military service. Additional criteria of eligibility were that the men and their mothers were born in the country where the men were currently living, and that the men had been born in 1979, 1980 or 1981.

The men were included and investigated in the same periods as the health authorities undertook the compulsory medical examination: for Denmark, in the spring, autumn and winter seasons; for Finland, in all four seasons; and for Estonia and Norway, in the winter and spring. The overall study period extended from March 1997 to January 2000. Within the individual centres the study periods were: Finland, from February 1998 to January 2000 (n = 324, participation rate 13%); Estonia, from December 1997 to June 1999 (n = 104, participation rate 19%); Norway, from January 1998 to April 1998 (n = 240, participation rate 17%); and Denmark, from March 1997 to March 1999 (n = 300, participation rate 19%). In total, 968 young men participated in the study. Results of 114 (38%) of the 300 Danish men have been published previously in part (Andersen et al., 2000Go), but as the men also fulfilled the inclusion criteria for the present study they were thus included.

In the following sections, country names are used for descriptions in order to avoid possible confusion between Turku and Tartu.

Data acquisition
In Denmark, Estonia and Norway the men received written information about the study on the day of the medical examination, and were offered further verbal information when the military health authorities had completed their examination. The men were permitted either to provide immediate acceptance to participate, or to forward their written acceptance by post at a later date. In Finland, the compulsory medical examinations were carried out at several smaller clinics, and lists of conscripts were provided by the military health authorities. Hence, all men were sent a letter inviting them to join the present study, with replies returned either by telephone or by post.

For all study sites, an appointment was made for attendance in the hospital/laboratory participating in the study for those men who had agreed to participate in the study. All participants were instructed to abstain from ejaculation for at least 48 h before attendance at the clinic. The current recommendation (World Health Organization, 1992Go) is that semen samples are collected after a minimum abstinence from ejaculation of 48 h, but not more than 7 days, in order to standardize the influence of this factor. In the present study, no upper time limit was given, as a reduction in the number of participants was anticipated if such a limit were to be introduced. On the day of attendance at the clinic, each man returned a completed questionnaire (see below), underwent a physical examination, and provided both blood and semen samples. Participants received financial compensation according to local custom for their participation.

Questionnaires
Prior to the study, a standardized questionnaire was developed in the English language and translated into Finnish, Estonian, Norwegian and Danish. These translated questionnaires were back-translated to control for translation errors. In order to assure the quality of the information regarding previous conditions, the questionnaire was sent to participants before their attendance at the hospital/laboratory, and they were asked to complete it (if possible) in collaboration with their parents. The questionnaire included information on age and previous or current diseases, including any known history of fertility.

Physical examination
A physical examination of each participant was performed on the day of delivery of his semen sample. An evaluation of the Tanner stages of pubic hair was performed. For the assessment of testicular size, all examiners used the same type of wooden orchidometer (Pharmacia & Upjohn, Denmark).

Blood samples
A blood sample was withdrawn from a cubital vein of each participant, centrifuged, and the serum was separated and frozen. Serum from participants in Finland, Estonia and Norway was sent frozen to Denmark for a centralized analysis in Department of Growth and Reproduction, Rigshospitalet, Denmark.

Serum levels of FSH, LH and sex hormone-binding globulin (SHBG) were determined using a time-resolved immunofluorometric assay (Delfia, Wallac, Turku, Finland). Testosterone levels were determined using a time-resolved flouroimmunoassay (Delfia, Wallac, Turku, Finland), estradiol by radioimmunoassay (Pantex, Santa Monica, CA, USA) and inhibin-B by a specific two-sided enzyme immunometric assay (Serotec, UK). Intra- and inter-assay coefficients of variation (CV) for measurements of both FSH and LH were 3 and 4.5% respectively. CV for testosterone and SGBG were <8 and <5% respectively. The intra- and inter-assay CV for estradiol and inhibin-B were 7.5 and 13%, and 15 and 18% respectively. The free testosterone index was calculated as (total testosteronex100)/SHBG.

Semen samples
The semen samples were obtained by masturbation and ejaculated into a clean collection tube in the privacy of a room adjacent to the laboratory. The semen samples were maintained at 37°C until taken for analysis.

The analysis of semen samples was performed according to WHO guidelines (World Health Organization, 1992Go), but were further specified following a study of inter-laboratory variation previously published by some of the involved centres (Jørgensen et al., 1997Go).

The period of ejaculation abstinence was calculated as the time between current and previous ejaculation as reported by the men.

Ejaculate volume was estimated by weighing the collection tube. Phase-contrast microscopy (positive phase-contrast optics) was used for the examination of fresh semen.

For the assessment of sperm motility, 10 µl of well-mixed semen was placed on a clean glass slide (which had been kept at 37°C) and covered with a 22x22 mm coverslip. The preparation was placed on the heating stage of a microscope (37°C), and immediately examined at x400 magnification. The sperm were classified as either motile (WHO motility classes A+B+C) or immotile (WHO motility class D), in order to report the percentage of motile sperm. The motility assessment was performed in duplicate and the average value was calculated for both samples.

For assessment of the sperm concentration, the samples were diluted in a solution of 0.6 mol/l NaHCO3 and 0.4% (v/v) formaldehyde in distilled water. The sperm concentration was assessed using a haemocytometer (Bürker-Türk chamber in Denmark, and Improved Neubauer chamber in Norway, Estonia and Finland). Only sperm with tails were counted.

Smears were prepared for morphological evaluation, Papanicolaou stained and finally sent to Finland for assessment of sperm morphology according to strict criteria (Menkveld et al., 1990Go). All morphology assessments were performed in random and blinded order by one of the authors (A.H.).

External quality control of sperm concentration assessment
All centres participated in an external quality control programme for sperm concentration assessment during the period December 1997 to May 1999. Briefly, each month five blinded samples were sent from the Danish laboratory to the other laboratories. Fresh samples from normal semen donors were preserved by addition of 10 µl of a 3 mol/l sodium azide solution per 1 ml of the ejaculate after liquefaction. Each centre received 600 µl of semen sent by mail in 1 ml cryotubes. Thus, all centres, including the Danish centre, performed counting according to their techniques, 4–8 days after the semen preparation. The results were reported to the Danish centre for statistical analysis.

Data recording
Standardized questionnaires, record forms for physical examination and semen analyses were labelled with ID-numbers. The information linking ID-numbers to personal data was kept separately at each centre in order to preserve confidentiality. Questionnaires, results of physical examination and results of semen analysis were sent to Denmark and entered into a centralized database. Additionally, the results of hormone analyses were also entered into the database.

Statistical analysis
Between-group differences in self-reported previous diseases and diseases detected during clinical examination, men's year of birth, year of investigation, season of investigation and hour of day for blood sampling were tested with a Pearson {chi}2-test. A Kruskal–Wallis test was used to test between-group differences in men's age, duration of abstinence and duration from ejaculation to assessment of motility.

The age of the man, his year of birth, year of participation in this study, season of year and duration of abstinence were evaluated as possible confounders for the semen parameters. For percentages of motile sperm the duration from ejaculation to assessment of motility was evaluated as an additional confounder. The hormone data were evaluated for the same possible confounders as the semen parameters in addition to hour of day of blood sampling. The effects of the possible confounders were tested in a multiple regression analysis. Additionally, interactions between factors were investigated.

In order to correct for the skewed distribution, semen volume, sperm concentration, total sperm counts and the hormone results were normalized by natural logarithmic transformation before analysis. Motility was logit-transformed before analysis.

Standard multivariate regression analyses were carried out to compare centres and estimate the general level of each centre for semen and hormone levels. First, semen volume, sperm concentration and total sperm count were adjusted for the duration of abstinence (see Results). Second, the quality control data were normalized by natural logarithmic transformation before a two-way analysis of variance that showed significant inter-laboratory differences, and these differences were estimated. Thereafter, these quality control results were used to adjust sperm concentration and total sperm count in order to present comparable levels. The Copenhagen laboratory was chosen as reference laboratory in order to make the results as comparable as possible with results of previous publications including results from the Danish laboratory (Bonde et al., 1998Go; Andersen et al., 2000Go; Jørgensen et al., 2001Go). Estimates of hormone levels were adjusted in the regression analyses for hour of day of blood sampling. Additionally, the effects of testis size, Tanner stage of pubic hair and hormone parameters were also evaluated in multivariate regression analyses. The final models were subjected to standard checks of the residuals.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Semen parameters, based upon the raw data obtained in each city, are summarized in Table IGo as `observed' values.


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Table I. Semen parameters of young men from four Northern European cities
 
In all four groups of men, increasing duration of abstinence had an increasing effect on semen volume, sperm concentration and total sperm count up to ~96 h (P < 0.0005 for all three parameters), whereafter no further effect of a longer abstinence period could be observed. Neither the age of the man, his year of birth, year of investigation or season of investigation had any confounding effects on these parameters. Multivariate regression analysis accounting for period of abstinence showed no difference between semen volumes of men from the four countries, whereas the sperm concentrations and total sperm counts differed significantly between the centres (all P < 0.0005) when the quality control results were not taken into account. The quality control study revealed a significant difference between the four participating laboratories regarding assessment of sperm concentration (P < 0.0005) (Table IIGo). Any possible linear trend in laboratory estimate levels over time was also investigated and found to be non-significant. In the final calculations of sperm concentration and total sperm counts, corrections for these inter-laboratory differences were additionally included in the estimates (Table IGo, `adjusted' values). The 95% confidence intervals (CI) presented in Table IGo refer to the `certainty' of the assessment of centre level. From these estimates it is apparent that men from Finland and Estonia had higher sperm concentration and total sperm count than men from Norway and Denmark. Statistically significant differences were shown for the differences Finland versus Denmark (95% CI for difference 1.08–1.59), Finland versus Norway (95% CI for difference 1.06–1.60), Estonia versus Denmark (95% CI for difference 1.07–1.79) and Estonia versus Norway (95% CI for differences 1.05–1.82), whereas the differences Estonia versus Finland and Norway versus Denmark were statistically non-significant (95% CI for differences 0.82–1.37 and 0.81–1.23 respectively). The relatively large CI are due to the inclusion of the standard error for the quality control results in the calculations. The adjusted results including 95% CI for each country are also shown for a subgroup entirely based on men who had not taken any medication during the previous 3 months before participation in the study, without known fertility and without any previous or current andrological diseases showing the same patterns as detected for all men.


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Table II. Inter-laboratory differences (%) in assessment of sperm concentration observed from quality control study
 
With regard to the frequencies of motile sperm and morphologically normal sperm, no effect of any of the confounders was apparent. Both parameters differed between the centres (P < 0.005). For motility, no quality control data were available, whereas all morphology smears were assessed in a blinded way by one person. Estimates of frequencies of motile sperm, frequencies of normal sperm and corresponding 95% CI were also calculated from a regression analysis in order to show results in a similar way in Table IGo as the other semen parameters. Men from Estonia were found to have the highest percentage of motile sperm, followed by men from Denmark, Finland and Norway in that order. Men from Finland and Estonia had the highest frequency of morphologically normal sperm. Table IGo also shows the total number of motile and morphologically normal sperm. Highest values were detected among men from Estonia and Finland. These figures were calculated from the total number of sperm and the frequencies of motile and normal sperm respectively.

Hour of day of blood sampling had a statistically significant effect as confounder on LH (P = 0.04), testosterone (P = 0.02) and free testosterone index (P < 0.005), all with highest hormone levels in the morning hours, and on SHBG (P = 0.005), with lowest hormone values in morning hours. For inhibin-B, the effect of hour of day of blood sampling was non-significant (P = 0.059), though highest levels of inhibin-B were apparent in the morning hours. Non-significant effects of hour of day of blood sampling as confounder were detected for FSH and estradiol. Due to these results and previous publications finding a diurnal rhythm in serum levels of reproductive hormones (Carlsen et al., 1999Go, and references therein), the hour of day of blood sampling was included as confounder in the further analysis of the hormone values. None of the other investigated possible confounders had any effect on the hormone parameters. The observed and the adjusted results of the hormone assessments are summarized in Table IIIGo. The adjusted hormone values are given as though the blood samples were drawn between 10:00 and 11:00. Men from Finland had the highest level of inhibin-B, whilst men from Estonia had the lowest level. In all four groups, regression coefficients were significantly positive for inhibin-B (P < 0.0005) and negative for FSH (P < 0.0005) when included in a regression model for sperm concentration and total sperm count. The effects of increasing inhibin-B and decreasing FSH were not statistically different between the centres. The men from Finland also had the highest levels of LH, testosterone and free testosterone index, whereas the Danish men had the second highest level of testosterone and free testosterone index.


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Table III. Serum hormone parameters of young men from four Northern European cities
 
Factors of possible confounding influence on semen parameters are summarized in Table IVGo. All of these factors differed significantly between the four study groups. However, only the duration of abstinence could be shown to possess a confounding influence on semen volume, sperm concentration and total sperm count as previously stated, and hour of the day of blood sampling an effect on the majority of hormones. In the Finnish centre an almost equal number of semen samples was delivered in all four seasons. A sub-analysis of the Finnish data did not reveal any significant confounding effect of season.


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Table IV. Possible confounders on semen and/or hormone parameters in study of young men from four Northern European countries
 
Self-reported, previous conditions of the young men from the four countries are summarized in Table VGo. Finnish men most frequently reported having had retracted testes that spontaneously descended (9.9%). Less than 1% of Finnish and Norwegian men had been treated for cryptorchidism, whereas 3.7% of Danish men and 3.9% of Estonian men had been treated for this condition. The frequencies of men in the subgroup who had not been affected by the conditions are also shown in Table VGo.


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Table V. Self-reported, conditions of young men from four Northern European countries obtained from self-administered questionnaire. Results are shown as frequencies (%) of number of men (n)
 
The main physical examiner in Estonia examined 100% of the Estonian study subjects, the Finnish main examiner 96% of the Finnish study subjects, the Norwegian 98% of Norwegian study subjects, and the Danish main examiner 96% of the Danish study subjects. The physical examination showed that the majority of men had reached an adult level regarding Tanner stage of pubic hair although regional differences were apparent: Finland 99.7%, Estonia 89.8%, Norway 77.8% and Denmark 100% (P < 0.0005 for difference between groups). Those men who had not reached Tanner stage 5 in pubic hair (adult level) all had stage 4. A sub-analysis of the effect of Tanner stage of pubic hair among the Norwegian men showed a significant difference in semen volumes between stage 4 and 5 (P = 0.01); men with stage 5 pubic hair had semen volume of 3.6 ml (95% CI 3.2–4.1 ml), whereas the men with stage 4 had 3.1 ml (95% CI 2.6–3.5 ml). There was no significant difference between these groups regarding sperm concentration, total sperm count, frequency of motile sperm or frequency of normal morphology.

The median testicular volumes (left/right testicles) were: Finland 20/23 ml, Estonia 22/23 ml, Norway 15/15 ml and Denmark 18/20 ml (P < 0.0005 for difference between the groups). Previously, an inter-observer variation in the results of the clinical andrological examination, including the four main physical examiners in the present study, was published (Carlsen et al., 2000Go), but this did not specify the levels of individual examiners. From these original data-sets it may now be estimated that the Finnish examiner estimated testis size as being larger than did the other examiners; 23 ml (median) versus 18, 19 and 20 ml for the Danish, Norwegian and Estonian examiners respectively. Regression coefficients were significantly positive for mean testis size when included in a regression model for sperm concentration, total sperm count and percentage of morphologically normal sperm (all P < 0.0005). The effect of a relative increase in testis size was the same in all centres (sperm concentration, total sperm count and frequency of morphologically normal sperm; all P = NS).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this co-ordinated cross-sectional study, differences were detected in both the quantitative and qualitative semen parameters between well-defined groups of young men from the general population of four Northern European cities. Not only had the Finnish and Estonian men higher sperm concentrations and total sperm counts, they also had higher frequencies of morphologically normal sperm than the Norwegian and Danish men. As all four groups of men were investigated according to the same protocol, and inter-laboratory differences in assessment of sperm concentration were controlled by an external quality control programme, the detected differences seemed to be real and not due to inter-laboratory differences. In a recent study of fertile men from Finland, Denmark, France and Scotland, a difference between Danish and Finnish men was shown, with highest sperm counts among Finnish men and lowest among Danish men (Jørgensen et al., 2001Go). Thus, the Finnish–Danish gradient regarding sperm counts seems to be present both among fertile men and young men not selected due to fertility. The sperm counts of young Danish men described here corroborated the findings reported previously that showed a low sperm count among young Danish men (Andersen et al., 2000Go).

The present results are interesting in relation to reported incidences in testicular cancer in Denmark, Norway, Finland and the Baltic countries, as they indicate an east–west gradient in the semen parameters parallel to the gradient in the incidence of testicular cancer in these countries. That is, the best semen quality was detected in the areas having the lowest risk of testicular cancer (incidence rate in Finland is 2.8 per 105 population, and in Estonia is 1.2 per 105) and the poorest semen quality in the Danish and Norwegian groups who are at the highest risk for testicular cancer (incidence rates in Denmark and Norway are 10.4 per 105 and 8.8 per 105 respectively) (Ferlay et al., 2001Go). Thus, the present findings lend support to the hypothesis that testicular cancer and impaired spermatogenesis sometimes may share aetiological factors.

Although low, incidence rates for testicular cancer have been increasing in Finland and Estonia during the recent years (Adami et al., 1994Go; Ferlay et al., 2001Go). The question is then whether the frequency of men with poor semen quality is also increasing in these countries. There seems to be insufficient retrospective data to answer the question with certainty. However, the sperm count of Finnish men was lower than could be expected from previously published studies. Two publications (Suominen and Vierula, 1993Go; Vierula et al., 1996Go) have reported that Finnish men during the years 1958–1992 had high and unchanged mean sperm concentrations of ~94–114x106/ml. The differences between these data and the results of the present study are impressive. Sperm concentrations were, in principle, assessed according to the same methods in these two retrospective studies and the present study. However, the validity of such comparisons is always hampered by the fact that they are based on retrospective data and study groups that are not completely comparable. As previously stated, only prospective studies will be able to indicate a possible time trend in semen qualities (Irvine, 1996Go).

Copenhagen and Oslo are both larger cities than Turku and Tartu. The semen results presented here are most likely not a reflection of this difference in city size, although this suggestion cannot be ruled out by the present investigation. In a previous study of young men from Denmark, men from the cities of Copenhagen and Aalborg were described as one group (Andersen et al., 2000Go) because they appeared to have semen qualities at equal levels. The number of inhabitants in Aalborg is ~160 000 and in Copenhagen ~500 000 (Danmarks Statistik, 2001Go); thus, city size does not appear to be an explanatory factor by itself.

The rising influence of increasing duration of abstinence up to ~96 h for semen volume, sperm concentration and total sperm count and no effect on frequency of motile sperm and frequency of morphologically normal forms is in agreement with findings in a recent study of fertile men in Finland, Denmark, France and Scotland (Jørgensen et al., 2001Go). Many publications do not report the influence of abstinence but merely mention that ejaculation abstinence was kept within a certain span of hours or days. However, it has been described that men with low sperm counts have increasing frequency of progressive motile sperm and increasing total number of sperm if abstinence was as long as 6–10 days (Magnus et al., 1991Go). Thus, the WHO recommendation of 2–7 days of abstinence (WHO, 1992Go) may be both too short and too narrow.

It was surprising that no seasonal variation was found in sperm quality. In a recent study on fertile men from some of the same countries a pronounced effect of season was found, with the highest sperm count during the winter season (Jørgensen et al., 2001Go). There is no clear explanation for these differences in observed seasonality. One possibility is that seasonality appears during ageing, and this should be investigated by re-examining the same group at a later date.

The differences in sperm counts and frequencies of morphologically normal sperm should most likely not be due to inter-laboratory differences. The assessments of sperm morphologies were performed in a random order by one person. Quality control samples were sent on a monthly basis for assessment of sperm concentration, and the results from this programme were used to calibrate the different laboratory levels to a comparable level. In contrast, the frequencies of motile sperm cannot be compared reliably between the four groups. The motility assessment was highly subjective, and there was no possibility of undertaking a quality control study of this parameter. Previously, the inter-laboratory variation for motility assessment was shown to be of significant importance, however (Jørgensen et al., 1997Go).

In as much as the detected levels of sperm parameters were regarded as real, the question arises of whether the studied cohorts of young men could be regarded as fully mature. This question cannot be answered unequivocally unless the men were to be re-examined later in their life. However, the majority of men had a fully mature stage of pubic hair, adult testis sizes, and an adult testosterone level and free testosterone index. Additionally, no influence of Tanner stage 4 pubic hair versus stage 5 was detected for sperm concentration, total sperm count, motility and morphology. Thus, the present data do not suggest that immaturity could explain these findings.

The examined men were considered as being representative of the normal population of young men of the cities in which they lived, despite the participation rate being quite low; indeed, this may have caused a selection of unknown direction and magnitude. However, the men had essentially no prior knowledge of their own fertility potential and, especially in the Danish and Norwegian groups, only one man had previously been diagnosed with a varicocele, although this had not been operated on. Therefore attention to possible fertility problems was unlikely to have affected their motivation to participate. As shown in Table IGo, the same geographical pattern in semen parameters was detected when the analysis was based only on men without any known andrological `conditions' as detailed in Table VGo. This indicates that the few men with known fertility and known previous or current diseases were not the reason for the detected geographical differences. The financial compensation received for participating in the study was unlikely to have led to the selection of men with reduced semen quality. In fact, if compensation had not been given it is most likely that men suffering from some kind of disease would be more interested in participating—in the hope of receiving advice—than men without diseases. Previously, in the Copenhagen centre, blood samples were collected only during a 2-week period from those men who attended the compulsory medical examination. The participation rate was 79%, and serum levels of sex hormones were comparable with those obtained from men at that centre who delivered semen samples (Andersen et al., 2000Go).

From a clinical viewpoint, the present study raises concern that the sperm concentration of both Norwegian and Danish men were close to a level at which a large proportion of men potentially may experience subfertility or infertility. Almost half of the Norwegian and Danish men had a sperm concentration which has been reported as being associated with increased time to pregnancy (TTP): one group (Bonde et al., 1998Go) related semen quality to fecundity, describing a decreasing TTP with increasing sperm concentration up to ~40x106/ml, while a more recent study of fertile men demonstrated a reduced TTP with increasing sperm concentration up to 55x106/ml (Slama et al., 2002Go).

Inhibin-B may represent the internal feedback to spermatogenesis in addition to feedback to pituitary FSH (Jensen et al., 1997Go; Anderson et al., 1998Go; von Eckardstein et al., 1999Go). Within all four groups of men, a correlation was found between increasing levels of inhibin-B and increasing sperm counts. However, inhibin-B levels could not be used to predict sperm counts between groups. The highest inhibin-B level was detected among Finnish men, as would be expected according to the sperm concentration and total sperm count. In contrast, the Estonian and Danish men had almost similar inhibin-B levels that were lower than those of the Norwegian men, despite the Estonian men having higher sperm counts. Thus, if spermatogenesis in the present study had been reflected only by inhibin-B values, a different conclusion would have been reached regarding the regional differences in semen quality. FSH did not add any further information as an indirect measure of semen quality.

The lower testicular volumes among the Danish and Norwegian men compared with Estonian and Finnish men may have been in accordance with the lower sperm counts among these men. However, the majority of differences in testicular volumes between countries were most likely a result of inter-observer variation, as this was of almost the same magnitude as the difference between the study subjects from the four countries (see Results). In all four centres increasing sperm concentration, total counts and frequency of normal sperm corresponded with increasing mean testis size.

In conclusion, young men from Estonia and Finland had higher sperm counts and higher frequencies of sperm with normal morphology compared with young men from Denmark and Norway, indicating an east–west gradient in the Nordic–Baltic area that was inverse to the gradient for the incidence of testicular cancer. Nevertheless, the semen quality levels detected for Finnish men were the lowest yet reported from Finland. These findings may have been due to genetic and/or environmental factors.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank all the volunteers who participated in the study; indeed, without their participation the study would not have been possible. This study was supported by the European Union (contract BMH4-CT96-0314), the Danish Research Council (grant no. 9700833), the Academy of Finland (research programme on Environmental Health), Turku University Central Hospital funds, Norwegian Research Council (grant no. 120912/310), Organon Agencies B.V (Estonia) and Estonian Science Foundation (grant no. 2991).


    Notes
 
7 To whom correspondence should be addressed at: Department of Growth and Reproduction, The Juliane Marie Centre, SectionGR-5064, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: njcph{at}rh.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 21, 2001; resubmitted on February 15, 2002; accepted on April 8, 2002.