1 Department of Growth and Reproduction, Rigshospitalet, section GR-5064, 9, Blegdamsvej, DK-2100 Copenhagen, 2 Institute of Community Health, Department of Environmental Medicine, Odense University, 3 Department of Urology, Aalborg Hospital and 4 Department of Biostatistics, University of Copenhagen, Denmark
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: general population/reproductive hormones/semen quality
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
So far clinical studies on semen quality have dealt with highly 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
; Van Waeleghem et al., 1996
) or infertility patients (MacLeod and Wang, 1979
; Bostofte et al., 1983
; Ombelet et al., 1996
; Berling and Wölner-Hanssen, 1997
; Andolz et al., 1999
) and may therefore be flawed by selection bias. Studies of semen quality, reproductive hormones and testicular size in unselected populations of young males have not previously been reported.
Due to the military drafting system in Denmark, all 18 year old men are required to attend a compulsory medical examination to determine their fitness for military service. In collaboration with the military health board, a unique opportunity was given to study important parameters of male reproductive health, including semen quality and reproductive hormones, in a quite unbiased population of young Danish men.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Group B
The relatively low participation rates obtained in group A might lead to selection bias. Therefore it was investigated whether the participants in group A were self-selected with regard to reproductive health or whether the low participation rates simply reflected a low motivation to participate in the rather demanding examination programme. Consequently, participants were enrolled according to a much less demanding protocol, which was expected to result in a higher participation rate and which would enable comparison of reproductive hormone concentrations as a measure of reproductive health. Subjects in this group (group B) were enrolled when they reported for their medical examination at the military health board during a 2 week period (November 1021, 1997). Inclusion criteria were the same as in group A. In this group the only request was for a blood sample drawn immediately after the medical examination was completed. Blood samples were drawn in a mobile laboratory placed just outside the building in which the military health board was located. Study subjects received approximately £30 for their participation. Altogether 195 men (79%) agreed to participate. Subsequently 12 of the participants from group B agreed to participate according to protocol A and were thus included in the 295 participants in the 199798 cohort of group A instead of in group B. Serum concentrations of follicle stimulating hormone (FSH), inhibin B, luteinizing hormone (LH) and testosterone were compared between group A and group B.
Semen analysis
In group A each man provided a semen sample that was obtained by masturbation into a wide mouthed plastic container in a room close to the semen laboratory. The period of abstinence was recorded and the semen sample was analysed according to the World Health Organization's guidelines (WHO, 1992) modified in accordance with Jørgensen et al. (1997). A minor change in the procedure of estimating the semen volume was introduced between the two group A cohorts. During the study of the first cohort, the semen volumes were estimated by weighing the plastic containers with semen samples and subsequently subtracting a standard weight of 16.1 g corresponding to a previously calculated mean weight of an empty plastic container. However, when it subsequently became clear that the weight of plastic containers varied from batch to batch (~0.5 g), all plastic containers used during the study of the second cohort were weighed before being handed out, and in this cohort the semen volumes were calculated as the weight difference between the filled plastic container and the empty container.
A previous study found inter-observer variability in semen analysis between different laboratories (Jørgensen et al., 1997), especially concerning motility assessments. In the present study only three technicians from our laboratory were involved in the semen analyses. Our technicians performed weekly intra-laboratory control by examining the same semen samples blindly and the between-technician variation in all semen parameters was less than 10% throughout the study period.
Hormone analysis
The concentrations of FSH, LH and testosterone were determined by time-resolved-immunofluorometric assay (DELFIA®, Wallac, Turku, Finland). Intra- and interassay coefficients of variation were below 8% in all DELFIA® assays. Inhibin B was determined using a specific enzyme immunometric assay, as described by Groome et al. (1996), with a detection limit of 18 pg/ml and intra- and interassay coefficients of 15 and 18% respectively.
Physical examination
All physical examinations were performed by three physicians (two in Copenhagen and one in Aalborg). Tanner stage of pubic hair and genital development, testicular volumes (determined by use of a Prader orchidometer), and the possible presence of a varicocele, a hydrocele or any genital malformation was recorded.
Questionnaire
Participants in group A each completed a questionnaire that was returned to the physician at the time of the physical examination. The questionnaire included information on previous history of fertility and/or genital diseases as well as other previous or current diseases. In addition, information was obtained on lifestyle factors such as smoking and drinking habits, as well as education and occupation. For participants in group B and for all non-participants, information on place and year of birth as well as education and occupation was obtained from the military health board.
Statistical analysis
Between-group differences in hormone concentrations, sperm motility, and period of abstinence were tested by the non-parametric MannWhitney test. The significance of between-group differences in sperm concentrations, semen volumes, and total sperm counts was tested by analysis of covariance controlling for period of abstinence. Analysis of covariance was performed on cubic root transformed values, as the cubic root transformation gave a normal distribution (sperm concentration and total sperm count) or near normal distribution (semen volume) of these parameters. Correlations between testis volume, semen parameters and hormone concentrations were tested by Spearman's rank correlation. All statistical calculations were performed in the statistics package for social sciences (SPSS) for windows, release 7.5.2.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
After controlling for period of abstinence, no difference was found in sperm concentration between men enrolled in 199697 and 199798 (see Table I). A minor difference in semen volume, and therefore in total sperm count, was observed between the two cohorts. However, this difference was most likely due to a slight change in the method of estimating the volume, as described in the methods section, which is supported by the fact that there were no differences in sperm motility or morphology. Furthermore, no differences were found in the concentrations of any of the reproductive hormones between the 199697 and 199798 cohorts except for a minor difference in FSH concentration (Table II
), a difference which is of no clinical relevance and may be explained by the long-term (12 year) variation in the assay. In addition, the FSH concentrations in group B and the second cohort of group A, which were analysed during the same period, were concordant whereas the FSH concentrations in the first cohort of group A, which was analysed 1.5 years earlier, showed a small (and similar) difference with regard to both group B and the second cohort of group A. When the two group A cohorts were combined, no significant differences in the concentrations of FSH or any of the other reproductive hormones were observed between men in group A and men in group B (see Table II
).
As the two cohorts of group A were very similar with regard to both semen parameters and reproductive hormones, they are described and discussed combined in the following. Among all men in group A 25 and 48% respectively had sperm concentrations below 20 x106/ml and 40 x106/ml. A total of 17% had less than 30% morphologically normal spermatozoa and 15% had less than 50% motile spermatozoa which are defined as normal reference values by WHO (WHO, 1992). WHO recommends a period of abstinence of at least 48 h (WHO, 1992
). A total of 521 participants in group A had a period of abstinence above 48 h and their median sperm concentration and median total sperm count were slightly higher than the general medians (45.0 x 106/ml and 132 x 106 respectively, see Table I
). Among the men with a period of abstinence above 48 h, 21% had sperm counts below 20 x106/ml and 43% had sperm counts below 40 x106/ml.
The following conditions may affect semen quality: current or previously treated varicocele, current or previously treated cryptorchidism, torsion of one or both testicles, inguinal hernia, epididymitis, genital infections, parotitis in adulthood, and testicular cancer. Altogether 29% of the men in group A had currently or had previously had one or more of these diseases (Table III). The median sperm concentration (44 x106/ml) and the median total sperm count (121.6 x106) among the men with no present or previous diseases in the reproductive organs were higher than among the men who had diseases in the reproductive organs (34 x106/ml and 74.5 x106). However, even among the men who had no diseases in the reproductive organs and had a period of abstinence above 48 h (n = 381), 18 and 40% respectively had sperm concentrations below 20 and 40 x106/ml.
The correlations between testicular volume, semen quality and reproductive hormones are summarized in Table IV.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important question to consider is whether selection bias may play a role, since less than 20% of the potential study populations participated in the semen studies. However, the place and year of birth of the participants as well as their educational status did not differ from the non-participants. Furthermore, it is believed that the participants are representative of the general population of 1820 year old men in Denmark for the following reasons: (i) the men had essentially no prior knowledge of their own fertility potential and therefore this is unlikely to have affected their motivation to participate; (ii) no differences in sperm counts and biomarkers of spermatogenesis (FSH and inhibin B) were found between the 199697 and 199798 cohorts; (iii) the serum concentrations of the reproductive hormones FSH, inhibin B, LH and testosterone found in those men who delivered semen samples did not differ from the concentrations found in the participants in group B, who only had a blood sample drawn and who it is believed represent the general population due to the high participation rate (79%). As serum concentrations of both FSH and inhibin B have previously been shown to reflect spermatogenesis (Jensen et al., 1997), this suggests that the spermatogenic potential observed in the present semen quality study can be considered to reflect the status in the general population of young men.
It might be argued that this group of young men does not represent mature adults. However, as many as 97% were fully virilized with normal adult pubic hair (at least Tanner stage 5), and they had testicular sizes within the normal range. Furthermore, in a previous study (Nielsen et al., 1986) the first occurrence of spermatozoa (spermarche) in the urine was found at a median age of 13.4 years and a median Tanner stage of 2.5. Other studies have shown that adult concentrations of reproductive hormones are established before the age of 18 years (Andersson et al., 1997
). In addition, preliminary results from a follow-up study of 150 of the men from the 199697 cohort, including semen analysis every 3 months, do not indicate any improvement in sperm concentration within a year (unpublished data). Thus, immaturity of the volunteers does not seem to explain the results.
The frequencies of current or previous genital diseases, including present varicocele (10%) and cryptorchidism with spontaneous descensus (8.8%), correspond well with published rates in the general Danish male population (Øster, 1971; Blom, 1984
; Møller et al., 1996
). This indicates that the poor semen quality is not due to a high rate of men with genital diseases.
A crucial question is whether the findings have relevance with respect to fertility. According to the WHO guidelines (WHO, 1992) a sperm concentration below 20 x106/ml is abnormal. However, these guidelines are not based on studies of fertility but are arbitrarily defined by a committee of international experts. Previous reference ranges for normal men were considerably more strict and delineated the lower range for sperm concentration at 60 x106/ml (MacLeod, 1946
), but also these reference ranges were arbitrarily defined. A recent study related semen quality to fecundity (waiting time to pregnancy of a couple) (Bonde et al., 1998b
) and a decreasing time to pregnancy was found with increasing sperm concentration up to approximately 40 x106/ml. It raises great concern that 40% of the men in the present study who had a period of ejaculation abstinence above 48 h, and who had never suffered from any genital diseases, fell below this value and were thus in the range of suboptimal semen quality. Even using the current WHO guidelines, a major fraction of these individuals (18%) would be classified as having oligozoospermia. In addition, a substantial number of the participants had a relatively high number of abnormal forms of spermatozoa as well as reduced motility. It is well known, though, that great inter-observer variability in sperm motility and morphology exists between different laboratories (Jørgensen et al., 1997
). In contrast, determination of sperm concentration based on the conventional haemocytometer, which was used in the present study, is a reliable method with acceptable intra- and inter-individual variation (Jørgensen et al., 1997
).
It is difficult to explain the low values of sperm count in young Danish men. A short period of abstinence may contribute to a low sperm count, but even when excluding those with a period of abstinence below 48 h, more than 40% of the men had a sperm concentration below 40 x106/ml. A significant correlation was also found between reproductive hormones, sperm count and testicular volume, indicating that low sperm counts were due to intrinsic biological factors rather than a short period of abstinence.
Seasonal variations in sperm concentrations, with the lowest values during the summer and peak values during late winter and spring months have previously been reported (Levine et al., 1988). Since the semen samples in the present study were collected during autumn, winter and spring seasons, the low sperm counts could not be ascribed to seasonal variation.
It remains to be seen whether these findings are generally applicable to populations of young men in the industrialized countries. Denmark seems to have relatively high rates of male reproductive abnormalities, including cryptorchidism (Ansell et al., 1992) and testicular cancer (Adami et al., 1994
; Forman and Møller, 1994
). The latter disease is possibly of fetal origin, and it has been postulated that an increased exposure to factors that inhibit the development of the fetal germ cells may be important for the pathogenesis of spermatogenic disorders and testicular cancer (Sharpe and Skakkebæk, 1993
). According to this hypothesis, changes in semen quality and frequency of testicular cancer should be related to year of birth. Previous studies (Auger et al., 1995
; Irvine et al., 1996
; Bergström et al., 1996
; Bonde et al., 1998a
) have in fact indicated that this might be the case, and the birth cohort effect of testicular cancer seems to be stronger than the time trend effect (Bergström et al., 1996
). Furthermore, in a Danish study including 1196 men from 10 cross-sectional occupational studies (Bonde et al., 1998a
), the median sperm concentration fell from 63 x106/ml among men born from 193749 to 52 x106/ml among men born from 197074. Sperm concentrations decreased by 1.24% per year after control for confounders. The present cohort born from 197679 seems to fit into this pattern with a median concentration of 41 x106/ml. In this connection it is interesting to compare the present findings with recent data on semen quality in men who were born approximately 10 years before the present group of participants. Firstly, in this group of men with a mean age of 28.2 years (SD 3.0) and with no knowledge of their fertility, the median sperm concentration was significantly higher (52 versus 41 x106/ml) and secondly, the concentration of inhibin B, a biomarker of spermatogenesis, was also significantly higher (median 202 versus 184 pg/ml) (Jensen et al., 1997
).
In conclusion, surprisingly low sperm counts were found in this study of young men. In fact, more than 40% of young adult Danish men have sperm counts below 40 x106/ml, which according to a recent study (Bonde et al., 1998b) is associated with decreased fertility. Thus, the findings indicate that a high proportion of young Danish men may experience reduced fertility. Possible causes for this high frequency of suboptimal semen quality are obscure and need to be explored. Also it remains to be seen whether these findings apply to young male populations in other industrialized countries. An international collaboration to address this question has been initiated.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersson, A-M., Juul, A., Petersen, J.H. et al. (1997) Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone testosterone and estradiol concentrations. J. Clin. Endocrinol. Metab., 82, 39763982.
Andolz, P., Bielse, M.A. and Vila, J. (1999) Evolution of semen quality in North-eastern Spain: a study in 22 759 infertile men over a 36 year period. Hum. Reprod., 14, 731735.
Ansell, P.E., Bennett, V., Bull, D. et al. (1992) Cryptorchidism: a prospective study of 7500 consecutive male births, 19848. Arch. Dis. Child., 67, 892899.[Abstract]
Auger, J., Kunstmann, J.M., Czyglik, F. et al. (1995) Decline in semen quality among fertile men in Paris during the past 20 years. N. Engl. J. Med., 332, 281285.
Bergström, R., Adami, H-O., Möhner, M. et al. (1996) Increase in testicular cancer incidence in six european countries: a birth cohort phenomenon. J. Natl Cancer Inst., 88, 727733.
Berling, S. and Wölner-Hanssen, P. (1997) No evidence of deteriorating semen quality among men in infertile relationships during the past decade: a study of males from Southern Sweden. Hum. Reprod., 12, 10021005.[ISI][Medline]
Blom, K. (1984) Undescended testis and the time of spontaneous descent in 2516 schoolboys. Ugeskr. Laeger, 146, 616617.[Medline]
Bonde, J.P.E., Jensen, T.K., Larsen., S.B. et al. (1998a) Year of birth and sperm count in ten Danish occupational studies. Scand. J. Work Environ. Health, 24, 407413.[ISI][Medline]
Bonde, J.P.E., Ernst, E., Jensen, T.K. et al. (1998b) Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet, 352, 11721177.[ISI][Medline]
Bostofte, E., Serup, J. and Rebbe, H. (1983) Has the fertility of Danish men declined through the years in terms of semen quality? A comparison of semen qualities between 1952 and 1972. Int. J. Fertil., 28, 9195.[ISI][Medline]
Bujan, L., Mansat, A., Pontonnier, F. et al. (1996) Time series analysis of sperm concentration in fertile men in Toulouse, France between 1977 and 1992. Brit. Med. J., 312, 471472.
Carlsen, E., Giwercman, A., Keiding, N. et al. (1992) Evidence for decreasing quality of semen during past 50 years. Brit. Med. J., 305, 609613.[ISI][Medline]
Comhaire, F.H., Vermeulen, L. and Schoonjans, F. (1987) Reassessment of the accuracy of traditional sperm characteristics and adenosine triphosphate (ATP) in estimating the fertilizing potential of human semen in vivo. Int. J. Androl., 10, 653662.[ISI][Medline]
Fisch, H., Goluboff, E.T., Olson, J.H. et al. (1996) Semen analyses in 1283 men from the United States over a 25-year period: no decline in quality. Fertil. Steril., 65, 10091014.[ISI][Medline]
Forman, D. and Møller, H. (1994) Testicular cancer. Cancer Surv., 19/20, 323341.
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1996) Measurement of dimeric Inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14011405.[Abstract]
Irvine, S., Cawood, E., Richardson, D. et al. (1996) Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years. Brit. Med. J., 312, 467471.
Jensen, T.K., Andersson, A-M., Hjollund, N.H.I. et al. (1997) Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone concentrations. A study of 349 Danish men. J. Clin. Endocrinol. Metab., 82, 40594064.
Jørgensen, N., Auger, J., Giwercman, A. et al. (1997) Semen analysis performed by different laboratory teams: an intervariation study. Int. J. Androl., 20, 201208.[ISI][Medline]
Lemcke, B., Behre, H.M. and Nieschlag, E. (1997) Frequently subnormal semen profiles of normal volunteers recruited over 17 years. Int. J. Androl., 20, 144152.[ISI][Medline]
Leto, S. and Frensilli, F.J. (1981) Changing parameters of donor semen. Fertil. Steril., 36, 766770.[ISI][Medline]
Levine, R.J., Brown, M.H., Bordson, B.I. et al. (1988) Deterioration of semen quality during summer in New Orleans. Fertil. Steril., 49, 900907.[ISI][Medline]
MacLeod, J. (1946) The semen specimen. Laboratory examination. In Engle, E.T. (ed.), Conference on Diagnosis in Sterility. Charles C. Thomas, Springfield, IL, pp. 315.
MacLeod, J. and Wang, Y. (1979) Male fertility potential in terms of semen quality: a review of the past, a study of the present. Fertil. Steril., 31, 103116.[ISI][Medline]
Møller, H., Prener, A. and Skakkebæk, N.E. (1996) Testicular cancer, cryptorchidism, inguinal hernia, testicular atrophy, and genital malformations: case-control studies in Denmark. Cancer Causes Contr., 7, 264274.[ISI][Medline]
Nielsen, C.T., Skakkebæk, N.E., Richardson, D.W. et al. (1986) Onset of the release of spermatozoa (spermarche) in boys in relation to age, testicular growth, pubic hair, and height. J. Clin. Endocrinol. Metab., 62, 532535.[Abstract]
Olsen, G.W., Bodner, K.M., Ramlow, J.M. et al. (1995) Have sperm counts been reduced 50 percent in 50 years? A statistical model revisited. Fertil. Steril., 63, 887893.[ISI][Medline]
Ombelet, W., Maes, M., Vandeput, H. et al. (1996) Chronobiological fluctuations in semen parameters with a constant abstinence period. Arch. Androl., 37, 9196.[ISI][Medline]
Paulsen, C.A., Berman, N.G. and Wang, C. (1996) Data from men in greater Seattle area reveals no downward trend in semen quality: further evidence that deterioration of semen quality is not geographically uniform. Fertil. Steril., 65, 10151020.[ISI][Medline]
Sharpe, R.M. and Skakkebæk, N.E. (1993) Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 341, 13921395.[ISI][Medline]
Sheriff, D.S. (1983) Setting standards of male fertility I. Semen analyses in 1500 patients a report. Andrologia, 15, 687692.[ISI][Medline]
Skakkebæk, N.E., Rajpert-De Meyts, E., Jørgensen, N. et al. (1998) Germ cell cancer and disorders of spermatogenesis: an environmental connection? APMIS, 106, 312.[ISI][Medline]
Suominen, J. and Vierula, M. (1993) Semen quality of Finnish men. Brit. Med. J., 306, 1579.[ISI][Medline]
Swan, S.H., Elkin, E.P. and Fenster, L. (1997) Have sperm densities declined? A reanalysis of global trend data. Environ. Health Perspect., 105, 12281232.[ISI][Medline]
Van Waeleghem, K., De Clercq, N., Vermeulen, L. et al. (1996) Deterioration of sperm quality in young healthy Belgian men. Hum. Reprod., 11, 325329.[Abstract]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. Cambridge University Press, Cambridge.
Øster, J. (1971) Varicocele in children and adolescents. Scand. J. Urol. Nephrol., 5, 2732.[Medline]
Submitted on August 20, 1999; accepted on October 26, 1999.