Higher sperm counts in Southern Sweden compared with Denmark

J. Richthoff1, L. Rylander4, L. Hagmar4, J. Malm2 and A. Giwercman3,5

1 Department of Urology and 2 Department of Clinical Chemistry, Malmö University Hospital, 3 Fertility Centre, Scanian Andrology Centre, Malmö University Hospital, SE 20502, Malmö and 4 Department of Occupational and Environmental Medicine, Lund University Hospital, SE 21885, Lund, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: A common aetiology behind time-related deterioration of male reproductive function, including decline of sperm concentration and rising incidence of testicular cancer and cryptorchidism, has been suggested. Accordingly, a high incidence of testicular cancer and low sperm counts have been observed in Denmark, while in Finland opposite figures were found. This may be due to genetic factors. The incidence of testicular cancer is 50% lower in Sweden than in Denmark. Data on sperm counts in a population of Danish military conscripts have been published and we wished therefore to compare them with semen parameters in a corresponding cohort from Southern Sweden, geographically and genetically very closely related to Denmark. METHODS: A total of 305 military conscripts was recruited and investigated in an identical way as those in the Danish study including a questionnaire, physical examination and semen analysis. RESULTS: Men born and raised in Sweden had a 23% higher mean sperm concentration, a 31% higher mean total sperm count and a 14% higher seminal volume compared with their Danish counterparts. CONCLUSIONS: The differences in reproductive parameters between the two genetically very similar populations could not be explained by possible confounders and may be due to unknown environmental or lifestyle-related factors.

Key words: environment/geographical trend/lifestyle/sperm concentration/testicular cancer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It has been suggested that human semen quality has deteriorated during the past few decades (Carlsen et al., 1992Go; Giwercman et al., 1993Go). This sign of impairment of male reproductive function has been aetiologically linked to increasing incidence of other abnormalities of male genital organs (Toppari et al., 1996Go). There is no doubt that the incidence of testicular germ cell cancer has increased by a factor of 2–3 worldwide during the past 4–5 decades (Adami et al., 1994Go; McKiernan et al., 1999Go). A possible rise in the frequency of congenital abnormalities such as cryptorchidism and hypospadias has also been suggested (Toppari et al., 1996Go).

Looking for a common cause to the above-mentioned signs of impairment of male reproductive function, focus has been directed toward environmentally derived compounds with hormone-like action, so-called endocrine disrupters. It has been proposed that these endocrine disrupters, which are thought to act in estrogen- or anti-androgen-like fashion, affect the male gonad in early fetal life (Sharpe and Skakkebæk, 1993Go; Toppari et al., 1996Go), causing the so-called testicular dysgenesis syndrome (Skakkebæk et al., 2001Go).

The hypothesis of an aetiological link between testicular cancer and abnormal sperm production was supported by the finding of a possible correlation between the risk of testicular malignancy and the level of sperm concentration in a given male population. The incidence of testicular neoplasm is subject to a significant variation, even between geographically and socially closely related areas. Thus, the risk among Danish men is 5 times as high as in Finland (Adami et al., 1994Go). The significantly higher sperm concentration in Finnish men as compared with Danes (Jensen et al., 2000Go; Jørgensen et al., 2001Go) has strengthened the hypothesis of a common cause of testicular cancer and impaired sperm production. However, the discrepancy between Danish and Finnish men may be due not only to environmental or lifestyle factors but also to genetic differences (Rosser et al., 2000Go; Krausz et al., 2001Go).

Recently, a Danish study of military conscripts showed a median sperm concentration of 41x106/ml (Andersen et al., 2000Go). The Danish study was performed in the urban areas of Copenhagen and Ålborg. By using an identical protocol, we performed a study of military conscripts from the area of Malmö, a city in Southern Sweden 20 km from Copenhagen. The incidence of testicular cancer in Denmark is almost twice as high as in Southern Sweden. Since these two populations are genetically very similar, a finding of higher sperm counts among the Swedish men would emphasize the importance of environmental and lifestyle-related factors for the male reproductive function.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects
Approximately 95% of all 18 year old Swedish men undergo a medical health examination prior to military service. Only men with serious chronic diseases are, excluded a priori from this conscript examination. Thus, the conscripts are close to the general population of young Swedish men. All 2255 men living <60 km from the city of Malmö who underwent the conscript examination May to December 2000 were asked to participate. Among those, 305 (13.5%) participated in the study, which took place at the Malmö University Hospital.

Every participant filled in a questionnaire, underwent physical examination and delivered a semen sample for which he received 500 SEK.

The local ethics committee approved the study.

Physical examination
One physician (J.R.) physically examined ~90% of all men whereas another examiner (A.G.) saw the remaining 10%. Both were experienced in andrological examination. The examination included assessment of body proportions and virilization as well as a careful genital investigation. The presence of varicocele was noted and graded 1 to 3. Genital malformations, hydrocele and previous genital surgery were recorded.

Questionnaire
All participants were asked to fill in a questionnaire at home. This questionnaire was translated from Danish and had previously been used in another study (Andersen et al., 2000Go). The men were asked for information about prenatal and postnatal factors that might influence their reproductive function (e.g. birth place, ethnic origin and congenital genital malformations).

Semen analysis
Each man provided a semen sample by masturbation into a wide-mouthed plastic container in a room at the laboratory. The weight of the empty plastic container was subtracted from the total weight to obtain the semen volume in ml. The measurements were done by use of a Sartorius® scale and the results expressed to two decimal places.

The semen sample was analysed according to published recommendations (World Health Organization, 1999Go). The men were asked to keep 48–72 h of abstinence but in each case the actual length of the period was recorded. Sperm concentration was assessed by use of a modified Neubauer chamber and positive displacement pipettes were used for proper dilution of the ejaculate. Only three laboratory assistants performed the analyses of the ejaculates and the inter-observer coefficient of variation (CV) was found to be 8.5% for concentration assessment.

Background characteristics
The subjects included in the present study were all aged between 18 and 21 years (mean 18.2). Their average body mass index was 22.6 and 84% of them were fully virilized (Table IGo). The mean length of the period of abstinence was 85.1 h. In 7.9%, the clinical examination revealed a varicocele. The frequencies of other, self-reported, genital pathologies are given in Table IGo. The proportion of men in the study living in the city of Malmö (250 000 inhabitants) was 24% whereas the remaining 76% were living in smaller communities or rural areas. The percentages of semen samples delivered during different seasons were: 21.1% summer (June 21–September 20); 51.3% autumn (September 21–December 20); 7.3% winter (December 21–March 20) and 20.3% spring (March 21–June 20).


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Table I. Comparison of background characteristics for the Swedish military conscripts and the previously published (Andersen et al., 2000Go) Danish data
 
For the comparison with the Danish study (Andersen et al., 2000Go), the statistical analysis was focused on the 248 individuals who were born and raised in Sweden.

The background characteristics, including the previously published Danish data (Andersen et al., 2000Go), are summarized in Table IGo.

Statistics
The influence of ethnic origin, place of living (urban versus rural), season when semen sampling was obtained, and categorized length of abstinence period (<48, 49–72, 73–96, 97–120, >120 h) on seminal volume, sperm concentration, and total sperm count, respectively, were analysed in general linear regression models. Furthermore, in order to minimise the effect of length of the abstinence period when comparing our results with those of Andersen et al. (2000), semen characteristics were evaluated separately in those subjects having at least 48 h of sexual abstinence prior to delivering the ejaculate.

The confidence intervals (CI) for the mean differences of seminal volume, sperm concentration, and total sperm count between the Swedish (including only those born and raised in Sweden) and the Danish conscripts were calculated based on a normal distribution (Altman, 1991Go). For comparison between the countries of the fractions with self-reported cryptorchidism, both total numbers and restricted to those who had been treated for testicular maldescent, Fisher's exact test was performed.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Own results
Seminal volume, sperm concentration and total sperm count for all 305 Swedish conscripts who participated in the study are presented in Table IIGo. Neither ethnic origin nor place of living significantly explains the variation of these outcome measures. There was no seasonal variation in any of the sperm characteristics. Length of abstinence period was, however, significantly associated with sperm concentration (P = 0.001) as well as with total sperm counts (P < 0.001). Restricting the analyses to the 248 conscripts born and raised in Sweden changed the results only marginally (Table IIGo). In the interval 24–96 h of abstinence, where a linear relation between time and sperm count was apparent, an increased abstinence period of 1 h corresponded to an increase in total count of 3.3x106 sperm (95% CI 1.9–4.6). In the group of subjects having abstinence period of >=48 h, the mean and median times of abstinence were 89 and 72 h respectively. Semen characteristics for this subgroup are given in Table IIIGo.


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Table II. Comparison of reproductive outcomes for the Swedish military conscripts and the previously published (Andersen et al., 2000Go) Danish data
 

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Table III. Comparison of semen parameters for the Swedish military conscripts and the previously published (Andersen et al., 2000Go) Danish data, restricted to the subjects with abstinence time >=48 h
 
The fraction of subjects with self-reported cryptorchidism was 3.9%. Including only those born and raised in Sweden decreased this fraction to 2.8%. For the proportions of men in whom testicular maldescent had been treated, the percentages were 1.3 and 1.2 respectively.

Comparison with the Danish data
The mean seminal volume was 14% (95% CI = 7–21%) higher among the subpopulation born and raised in Sweden compared with the Danish men (Table IIGo). The Swedish men also had a 23% (95% CI = 3–40%) higher sperm concentration and a 31% (95% CI = 14–47%) higher total sperm count, as compared with the Danish men. These differences remained statistically significant even when the analysis was restricted to those individuals having an abstinence period of >=48 h (Table IIIGo), although the mean length of the abstinence time was slightly lower among the Swedish males (89 versus 101 h). Compared with the Danish subjects, a significantly lower proportion of Swedish men reported cryptorchidism (2.8 versus 12.6%). However, when focusing only on those in whom this condition had been treated, the difference between the two cohorts was only borderline significant (1.2 versus 3.8%, P = 0.05). For the other variables that might affect testicular functions (Table IGo), the values were too small for meaningful comparisons.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We found significantly higher seminal volume, sperm concentration and total sperm count among military conscripts in Southern Sweden as compared with previously published data for a corresponding group of Danish men (Andersen et al., 2000Go). The total number of sperms was as much as 31% higher than in Denmark. The age-standardized incidence of testicular germ cell cancer in Denmark is 9.5 per 100 000 whereas in Sweden this figure is 4.4 per 100 000 (Ferlay et al., 1999Go). In Southern Sweden the risk of TGCC does not significantly diverge from the average of the country. Therefore, our findings may indicate that in this region of Sweden, lower incidence of TGCC, as compared with Denmark, is associated with higher sperm counts among young males. Such an association between the incidence of testicular malignancy and sperm production in a given population fits with the hypothesis of common cause of a range of disorders in the male reproductive tract (Sharpe and Skakkebæk, 1993Go; Skakkebæk et al., 2001Go).

Factors including inter-laboratory variation in assessment of semen parameters, selection bias, the length of abstinence period and season at time of sampling are important for interpretation of semen data and it needs to be discussed whether they can explain the observed difference between young Swedish and Danish men. In both countries the criteria for going through a conscript examination are very similar, and nearly 95% of all men undergo such examination. Among the Swedish men, 13.5% agreed to deliver a semen sample whereas 17.6% of the Danish men did so. It can be expected that men in the age group 18–19 years have no knowledge about their reproductive capability. The low participation rate should, therefore, not imply any selection bias with respect to fertility. Accordingly, based on the levels of reproductive hormones in participants and non-participants in the semen study, Andersen et al. concluded that the participants seemed to be representative for the whole groups of conscripts (Andersen et al., 2000Go). Therefore, we believe that the group of men delivering a semen sample was representative for the total cohort of military conscripts, at least with regard to their reproductive function. Furthermore, we found an identical mean BMI for the group of men delivering a semen sample, as is true for the whole group of Swedish conscripts.

The even distribution of prevalence of genetic abnormalities—which may affect semen quality without being linked to prenatal hormonal exposure—found in the Danish and the Swedish cohorts, is in agreement with a comparison of the two populations with respect to selection of subjects.

Most of the Danish men originated from an urban area, whereas a significant proportion of the Swedish subjects came from smaller communities or rural areas. We did not, however, find any significant difference in the sperm parameters between men living in the city of Malmö and men living in surrounding communities.

The Swedish men were on average 1 year younger than their Danish counterparts. The maturation of the male reproductive system may not be completed at the age of 18 years and the sperm production is, therefore, expected to remain unchanged or even increase during the following year of life. It should therefore rather imply lower but not higher sperm counts in 18 year old men as compared with those 1 year older. Birth cohort effect in relation to the male reproductive function has been postulated (Auger et al., 1995Go; Irvine et al., 1996Go). The Swedish conscripts were born 3–5 years later than their Danish counterparts. In view of the reports of a negative, time-related trend in sperm production (Giwercman et al., 1993Go), this difference should tend to give lower and not higher sperm counts among the Swedish conscripts.

The median abstinence period was 4.5 h longer in the present study, which might add to slightly higher sperm concentration and total sperm count in Swedish conscripts. Our calculations have, however, indicated that this discrepancy can only explain 30% (15x106) of the observed difference in total sperm count between the two countries. In accordance with this estimate, when the comparison was restricted to those subjects having an abstinence period of >=48 h, the differences in semen characteristics remained statistically significant, although the mean abstinence time was, for this subgroup, slightly shorter among the Swedish male than in their Danish counterparts.

We found no seasonal variations in any of the semen parameters although the proportion of samples collected during the winter period was rather low.

An inter-laboratory CV of ~30% was found for determination of sperm concentration in external quality control programmes based on a single mailing of semen aliquots (Neuwinger et al., 1990Go). It can be claimed that our results might be due to a systematic difference in sperm counting techniques between the two studies. No comparison between the laboratories in Copenhagen and in Malmö was performed in parallel with the sampling of ejaculates. Both semen laboratories were—during the periods of sample collection—supervised by the same person (A.G.). Furthermore, they strictly followed the World Health Organization recommendations including the use of positive displacement pipettes and haemocytometers. A longitudinal, external quality control programme with participation of six Nordic–Baltic laboratories and including monthly mailing of five semen aliquots with addition of preservative, has shown 50% reduction of the CV, when the sperm counting was made on fresh ejaculates, and not on mailed samples. Additionally, the relative counting level for any given laboratory—in relation to the other participants in the study—seemed to change over time. We found an inter-laboratory CV (9.1% for means and 9.7% for medians) when sperm concentration assessments, made by the six laboratories, were compared over a 1 year period (A.Giwercman et al., unpublished results). These figures were based on mailed aliquots and an even smaller degree of variation may be expected if fresh samples were analysed. When considering the assessment of seminal volume, in both centres, weighting of the container—without and with the ejaculate—was used for determination of the volume. Thus, it seems unlikely that methodological differences can account for the 14% higher ejaculate volume and 23% higher sperm concentration found in the current study. Therefore, we cannot exclude that bias factors related to the recruitment of subjects, or that laboratory methods, might add to the difference in semen parameters between the two populations. However, summarizing the impact of the most obvious confounders (Table IVGo), their effect should rather imply higher seminal volume and/or sperm concentration in the Danish cohort of military conscripts and not the reverse, as we found.


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Table IV. The expected impact of potential confounders on the difference between Denmark and Sweden in semen characteristics
 
The 12.6% frequency of self-reported cryptorchidism in the Danish study seems rather high. However, a 7% prevalence of cryptorchidism was found among >2500 schoolboys from the area of Copenhagen (Blom, 1984Go). The fact that the self-reported prevalence of cryptorchidism was much higher in one other study (Andersen et al., 2000Go) may be due to a real difference between the two populations. However, we found only a borderline significant difference between the two cohorts when the cases in which the retentio testis had been treated were calculated.

Regional differences in sperm concentration reflecting the discrepancy in the incidence of TGCC have previously been reported for the Danish and the Finnish populations (Jensen et al., 2000Go). The status of male reproductive function may be a result of interplay between genetic, environmental and lifestyle-related factors. Whereas there is an obvious genetic diversity between the Danish and Finnish men, including the Y chromosome (Rosser et al., 2000Go), which apparently plays an important role for the function of male reproductive organs (Krausz et al., 2001Go), the populations of Southern Sweden and Denmark were found to be very similar from a genetic point of view (Luca Cavalli-Sforza et al., 1994) including the prevalence of haplotype 16 of the Y chromosome (L.Beckman, personal communication). Therefore, the Danish–Finnish difference in reproductive parameters can, at least partly, be due to genetic factors, whereas, the indices of better male reproductive health found in Southern Sweden are most likely related to the impact of environmental and/or lifestyle factors. The exact nature of these factors is unknown and the design of the present study does not allow them to be identified. It has been suggested that sperm production, TGCC as well as cryptorchidism, are—as a part of testicular dysgenesis syndrome (Skakkebæk et al., 2001Go)—all related to exposure to endocrine disrupters acting in the early fetal life (Sharpe and Skakkebæk, 1993Go). Thus, we have to turn our attention to factors operating in the early 1980s and it seems reasonable to consider differences in food consumption habits and food processing as some of the candidate risk factors.

In conclusion, we found significantly higher seminal volume, sperm concentration and total sperm count in young men from Southern Sweden as compared with young Danish men. These figures fit with 50% lower incidence of TGCC in Southern Sweden (Ferlay et al., 1999Go) as compared with Denmark. Since the two populations are presumed to be genetically very similar, our results might be due to inter-country difference in environmental and lifestyle-related factors with possible impact on male reproductive function. However, further studies, based on new cohorts of Danish and Swedish males and using additional markers of reproductive function, e.g. inhibin B, are warranted in order to confirm the findings of current report.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Erna Stridsberg, Rita Hofvander and Dr Göran Svensson for their help in recruiting the participants in the study. Anna Bremer, Camilla Anderberg and Katarina Jepson are thanked for their technical assistance. Professor Lars Beckman contributed with valuable discussion on genetic trends in the Nordic area. Associate Professor Jan Schönebeck is thanked for his enthusiastic support. The study was supported financially by Swedish governmental funding for clinical research, Crafoordska Fund, Ove Tulefjords Fund and Foundation for Urological research.


    Notes
 
5 To whom correspondence should be addressed. E-mail: aleksander.giwercman{at}kir.mas.lu.se Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 13, 2001; resubmitted on March 5, 2002; accepted on May 2, 2002.