1 Department of Growth and Reproduction, Rigshospitalet, The Juliane Marie Centre, Copenhagen, Denmark, 2 Université Paris V, Groupe Hospitalier Cochin, Paris, France, 3 MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK, 4 University of Turku, Institute of Biomedicine, Turku, Finland, 5 Department of Biostatistics, University of Copenhagen, Copenhagen, and 6 The Fertility Clinic, Rigshospitalet, Copenhagen, Denmark
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Abstract |
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Key words: fertile men/reference group/regional differences/semen quality
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Introduction |
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The regional differences and adverse trends seen in male reproductive health clearly involve aspects other than spermatogenesis. It is generally agreed that the incidence of testicular germ cell cancer in adults is increasing and also shows great geographical variation (Adami et al., 1994; Forman and Møller, 1994
). Furthermore, congenital malformations of the male genital tract, such as hypospadias and cryptorchidism may have increased in some geographical regions (World Health Organization, 1991
; Ansell et al., 1992
), although valid data are only available from very few areas.
The controversies of much of the published data are in part due to the fact that previous clinical studies on semen quality have dealt with selected groups of men: volunteers enrolled after advertisement (Irvine et al., 1996; Paulsen et al., 1996
; Lemcke et al., 1997
), candidates for vasectomy (Sheriff, 1983
; Fisch et al., 1996
), semen donor candidates (Leto and Frensilli, 1981
; Auger et al., 1995
; Bujan et al., 1996
) or infertility patients (MacLeod and Wang, 1979
). In many studies, historical data collected for other purposes have been used without close attention to confounding factors which would be relevant to an analysis of secular or geographical trends. In most comparisons, the periods of abstinence from ejaculation, age and inter-laboratory differences have not been accounted for, to some extent because the underlying data were not obvious from the original reports. Although the present literature may suggest temporal and spatial differences in male reproductive health, it has also been pointed out that definitive evidence will only be provided by prospective studies (Irvine, 1996
; Swan et al., 1997
; Skakkebæk et al., 1998
). Moreover, because prospective studies on secular trends in male reproductive health will be of a long-term nature, study of the apparent regional differences (if confirmed) could provide clues to the aetiology of the problem.
Previous data have indicated that Danish men may have low sperm counts (Bostofte et al., 1983) compared with Finnish men, who have high and unchanged sperm counts (Vierula et al., 1996
). Furthermore, the sperm counts of French and Scottish men seem to have declined in recent years (Auger et al., 1995
; Irvine et al., 1996b). Therefore, a cross-sectional study was undertaken focusing on the possible geographical differences in semen quality, by studying the male partners of pregnant women from Denmark (Copenhagen), France (Paris), Scotland (Edinburgh) and Finland (Turku) using co-ordinated standardized investigation procedures. Male partners of pregnant women were chosen as study subjects because they appeared to constitute well-defined, demographically comparable groups in each of the participating countries. Inclusion of infertile men would lead to less well-defined study groups which could not be compared reasonably. However, in countries having a military drafting system as in Denmark it is possible to investigate men from the general population (Andersen et al., 2000
).
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Materials and methods |
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The eligibility criteria for each man were: 20 to 45 years of age at the time of invitation, residing in the local referral area of the hospital to which he was recruited, and being born in the country in which he was currently living. Further, the current pregnancy of the female partner had to be achieved by normal sexual relations, and not as a result of any treatment for subfertility or infertility (hormonal treatment, insemination, IVF or intracytoplasmic sperm injection, etc.). Participation in the study was accepted even if the man had a past history of cryptorchidism, orchitis, epididymitis, surgery of the genital tract (including varicocelectomy), chemotherapy, radiotherapy or other diseases which may affect reproduction. Chronic illness, previous treatment for infertility or subfertility, unwanted pregnancy or prolonged waiting time to pregnancy were not exclusion criteria.
Couples fulfilling the eligibility were asked to participate in the study. Altogether, 1082 men participated in the study; 349 (participation rate 43%) from Copenhagen, 207 (participation rate 15%) from Paris, 275 (participation rate 19%) from Turku and 251 from Edinburgh. The inclusion process (the only one possible) in Edinburgh did not allow for calculation of a participation rate. Except in Paris, the participants received financial compensation for their travel expenses, and/or lost working hours, according to local custom and practice within this field. Information on age and previous diseases of the study population is summarized in Table I.
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Questionnaires
On the day of attendance the men returned a completed standardized questionnaire which they had received in advance. The questionnaire included information on age, previous or current diseases and the number of previous pregnancies. Prior to the study, the standard questionnaire had been developed in English, and translated into Danish, Finnish and French. These translated questionnaires were back-translated to control for translation errors.
Semen samples
The semen samples were obtained by masturbation and ejaculated into a clean collection tube. WHO recommend that semen samples should be collected after a minimum of 48 h but not more than 7 days of ejaculation abstinence to standardize the influence this factor (World Health Organization, 1992). In this study, all men were asked to abstain from ejaculation for at least 48 h, but were not given any upper limit, as a reduction in the number of participants was anticipated if such a limit were to be imposed upon this group of partners of pregnant women. The period of ejaculation abstinence was calculated as the time between current and previous ejaculation as reported by the men.
In Paris and Edinburgh the semen samples were collected in the privacy of a room near the laboratories. Due to facility reasons, ~20% of samples from Turku and ~80% of samples from Copenhagen were collected at the men's home and delivered to the laboratory. If collected at home, the samples were protected from extremes of temperature (<20°C and >37°C) during transport to the laboratory. In the laboratories, the semen samples were kept at 37°C until analysed.
The analysis of semen samples was performed according to WHO conditions (World Health Organization, 1992), but further specified following assessment of inter-laboratory variation prior to the study (Jørgensen et al., 1997
). Ejaculate volume was estimated by weighing the collection tube with the semen sample and subsequently subtracting the predetermined weight of the empty tube, and assuming that 1 ml of ejaculate weighs 1 g. 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 a total magnification of x400. The microscope field was scanned systematically, and the spermatozoa were classified as either motile (WHO motility classes A+B+C) or immotile (WHO motility class D), in order to report the proportion of motile spermatozoa. The motility assessment was repeated on a second 10 µl aliquot of semen, and the average value was calculated for both samples.
For the assessment of sperm concentration, each semen sample was thoroughly mixed for at least 10 min in a rotation device. An aliquot of the sample was put into the diluent using a positive displacement pipette and mixed for a further 10 min. The diluent consisted of 50 g NaHCO3, 10 ml 40% formaldehyde and distilled water up to 1 litre. The sperm concentration was assessed using haemocytometers (Bürker-Türk chamber in Copenhagen and Turku; Neubauer chamber in Edinburgh; Thoma chamber in Paris). One drop of the diluted specimen was transferred to each chamber of the haemocytometers, which were allowed to stand for 5 min in a humid chamber before the cells were counted at a total microscope magnification of x400. Only spermatozoa with tails were counted.
Smears for morphology evaluation were made. The thickness of the smears was varied according to the sperm concentration in each sample. The smears were air-dried, fixed for 1 h with a mixture of absolute alcohol (2/3, v/v) and acetic acid (1/3, v/v), and then sent to Paris for a modified Shorr stain (World Health Organization, 1992), and assessment of sperm morphology according to criteria described previously (David et al., 1975
).
External quality control of sperm concentration assessment
All centres participated in an external quality control (QC) programme for sperm concentration assessment in the period January 1997 to June 1998. Briefly, each month five blinded samples were sent from the Copenhagen centre to the other laboratories. Fresh samples from normal semen donors were preserved by addition of 10 µl of a 3 mol/l (~20%) sodium azide solution per 1 ml of the ejaculate (after liquefaction). This procedure was used in order to obtain undiluted samples, since the dilution step is considered to be an important source of variation when sperm counting is performed. Each centre received 600 µl of semen. To obtain a sufficient volume, ejaculates from two or more donors were mixed, and the samples were sent by mail in 1 ml cryotubes. Thus, all centres performed sperm counting according to their techniques described above, 27 days after the semen preparation was performed, including Copenhagen. The results were reported to the Copenhagen centre for statistical analysis.
Physical examination
Physical examination of each participant was performed on the day of the delivery of his semen sample. Evaluations of testes disposition, varicocele and Tanner stages of pubic hair were performed with the men in standing position. For assessment of testis size all examiners used the same type of wooden orchidometer (Pharmacia & Upjohn, Denmark).
Data acquisition
Standardized questionnaires, record forms for physical examination and semen analyses were labelled with identification (ID) numbers. The information linking ID-numbers to personal data were kept separately in each centre so as to preserve confidentiality. Information from the questionnaires, and results of semen analysis and physical examination were sent to Copenhagen and entered into a centralized database.
Statistical analysis
Sperm concentrations and total sperm counts were best normalized by cubic root transformation before analysis to correct for the markedly skewed distribution. Multivariate regression analyses were carried out to compare differences between centres. In these analyses the general level of each centre was estimated while adjusting for known confounders, including age, abstinence time and season. Age and abstinence time entered the model as piecewise linear functions (linear splines); for example, one straight line for abstinence below 48 h, another straight line for abstinence periods 4896 h, etc. Season entered the model as a categorical variable allowing each of four seasons to have a different level (spring, MarchMay; summer, JuneAugust; autumn, SeptemberNovember; winter, DecemberFebruary). The final models were subjected to standard checks of the residuals. Natural logarithmic transformation gave models in which centre differences and effects of covariates are more easily interpretable. This alternative model approximates very closely the model obtained transforming with the cubic root and is used when reporting centre differences (see Table IV). The percentages of motile spermatozoa were logit-transformed and analysed in a multivariate regression model while adjusting for age, abstinence time, seasonal variation, and the delay from time of ejaculation to assessment of motility. The percentages of morphologically normal forms were arcsine-square root-transformed and also analysed in a multivariate regression model adjusted for age, abstinence time and seasonal variation.
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Results |
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The delay from time of ejaculation to assessment of motility was as follows (mean/median): Copenhagen 83/80 min; Paris 37/35 min; Edinburgh 36/35 min; and Turku 42/40 min (P < 0.0005).
The results of the quality control study of sperm concentration showed no significant differences between the four centres (P = 0.17). Additionally, no significant differences were detected when comparing the centres pair-wise (Table III). Therefore, no correction for laboratory differences was included in the statistical analysis.
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Table V shows, for each of the four cities, the expected calculated semen quality of a recently proven fertile, 30-year-old man, having an ejaculation abstinence period of 96 h. Examples for both the winter and summer seasons are given; these values are constructed based on the results from the regression analyses after taking the confounders into account.
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The parity (i.e. whether the men were involved in a pregnancy for the first time versus subsequent pregnancies) differed between the four investigated groups of men (P < 0.0005), as those involved in a pregnancy for the first time were as follows: Copenhagen 46%; Paris 37%; Edinburgh 71%; and Turku 48% (see Table I). However, no difference in sperm counts between those involved in first versus subsequent pregnancies were found (e.g. P = 0.65, for sperm concentration).
Self-reported, previous subfertility (waiting time to pregnancy of more than 1 year), treatment for infertility, cryptorchidism, testicular cancer, varicocele, testicular torsion, inguinal hernia, epididymitis, orchitis, gonorrhoea, diabetes and thyroid diseases did not differ between the groups of men from the four cities (see Table I). However, a high percentage of Finnish (12.4%) and Danish (16.0%) men reported a previous chlamydial infection compared with French (5.3%) and Scottish (2.0%) men (P < 0.0005). Within-centre comparisons did not reveal any differences between those reporting previous Chlamydia infection and those who had not had Chlamydia regarding period of ejaculation abstinence, semen volume, sperm concentration, total sperm count, proportion of motile spermatozoa and morphologically normal spermatozoa. Detailed information on how the diagnosis was established or on treatment was not available.
Physical examinations showed that none of the participants suffered from severe genital abnormalities, e.g. micro-penis or micro-testis, and all men (except for one from Copenhagen and three from Edinburgh) had both testes in the scrotum. The testicular volumes (mean of left and right testicles) were (median): Denmark 23.5 ml; Edinburgh 23.0 ml; France 22.5 ml; and Finland 23.0 ml. In all four groups of men, the right testis appeared to be larger than the left testis.
All Danish and Finnish men, 98.4% of Scottish men and 89.1% of French men were found to have an adult pubic hair distribution (i.e. Tanner stage 5). Differences in the number of men with varicoceles were detected at the physical examination for the four groups of men: Copenhagen 1%; Paris 4%; Edinburgh 5%; and Turku 3%.
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Discussion |
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Based upon the results, it was possible to calculate expected values for a standardized 30-year-old fertile man from each of the four cities. In Table V, the confidence intervals of the estimates are given in order to state the `certainty' of the given levels. These confidence intervals should not be interpreted as 95% confidence intervals for the populations.
The magnitude of the seasonal variations in sperm counts emphasized the importance of controlling for this factor. An apparent difference in sperm counts of ~30% occurred from winter to summer in all four centres. Some previous studies have also detected seasonal variations, and consistently the lowest sperm counts were detected during the summer season and highest during either autumn or winter seasons (MacLeod and Heim, 1945; Tjoa et al., 1982
; Spira, 1984
; Maier et al., 1985
; Gyllenborg et al., 1999
). These studies were performed either in Europe or the USA, and some included men of known fertility while others included men of known subfertility. However, other studies have been unable to detect seasonal variation. For example, an Australian study of `normal' men (Mallidis et al., 1991
) and a BelgianSouth African study of infertile men (Ombelet et al., 1966
) did not detect any seasonal variation. The present study was cross-sectional in nature, as were the majority of the previous published studies regarding seasonality. The possible seasonal variation should be investigated by longitudinal studies. Until convincing results of such studies are published, it is recommended that seasonality be included equally as other confounders such as abstinence period and age.
The differences in the proportions of motile and morphologically normal spermatozoa between men from the four cities gave a different ranking than the sperm counts, but the majority of differences in these parameters were not statistically significant. In spite of attempts to standardize motility assessments between laboratories, the inter-technician variation may still be considerable and may account for some of the differences. For the morphological assessment, staining and evaluation was centralized, and thus the results are not likely to be explained by technical reasons. Nevertheless, calculating the total number of morphologically normal forms per ejaculate revealed the same ranking as the sperm counts: Danish men had the lowest number, followed by men from Edinburgh and France, while Finnish men had the highest absolute number of normal forms.
It is interesting to consider our finding of geographical differences and previous reports on time trends in semen quality in connection with similar patterns in testicular cancer. The incidence of this disease is rising in almost all countries, and is five times higher among Danish men than among Finnish men (Adami et al., 1994; Forman and Møller, 1994
), who correspondingly, in the present study, had a much better sperm count. The adverse relationship between sperm count and the risk of testicular cancer is not only apparent in cohort studies, but is also seen in individuals (Møller and Skakkebæk, 1999
). The synchronized trends in semen quality and testicular cancer may reflect common prenatal risk factors (Skakkebæk et al., 1987
, 1998
; Møller and Skakkebæk, 1999
). It is generally accepted that the precursor cells of testis cancerthe carcinoma in-situ germ cellshave several characteristics of fetal germ cells, including stem cell markers (Skakkebæk et al., 1987
; Damjanov, 1991
; Jørgensen et al., 1995
; Rajpert-De Meyts et al., 1998
). They are thought to arise perinatally as a result of a carcinogenic change of the primordial germ cells. Also, epidemiological studies support the hypothesis of a fetal origin: both testicular cancer and semen quality have been linked to birth cohort effects. Thus, men born in Scandinavia during the Second World War had a relatively lower risk of developing testicular cancer in adult life than men born before or after the war (Møller, 1993
; Adami et al., 1994
). In addition, two studies have indicated that sperm counts seem to decline with a more recent year of birth (Skakkebæk et al., 1987
; Irvine et al., 1996
). A possible theory is that exogenous factors which interfere with the function and multiplication of the fetal Sertoli cell may be to blame for a syndrome of reduced sperm count, hypospadias, undescended testis and testicular cancer (Sharpe and Skakkebæk, 1993
; Bergman et al., 1996
). In this respect, it is noteworthy that the gradient in the incidence of hypospadias between Denmark and Finland seems to parallel that of testicular cancer (Toppari et al., 1996
).
In conclusion, this international coordinated study of fertile men has revealed significant differences in semen quality between men from four European cities. Although genetic differences cannot entirely be ruled out as causes, a more likely explanation for these findings seems to be differences in lifestyle or other environmental factors. Recently, endocrine disrupters have been in focus as possible aetiological, environmental factors. Whatever the causes are, the differences in male reproductive health between countries in Europe are considerable, and the fact that several aspects of reproductive function are involved (sperm production, testicular cancer, hypospadias) suggests that the differences are robust. Considering the great importance of reproductive health, these results should be followed by a close look at possible environmental and lifestyle differences between countries with the greatest differences in male reproductive health.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 12, 2000; accepted on January 26, 2001.