1 Laboratorio Investigaciones Seminológicas, Aribau 280, 08006 Barcelona, and 2 Departament Epidemiologia, Institut Municipal d'Investigació Mèdica, Dr. Aiguader 80, 08003 Barcelona, Spain
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Abstract |
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Key words: Semen analysis/semen quality/time-related change
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Introduction |
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The controversy over sperm counts began with a meta-analysis by Carlsen et al. (1992) which showed an important decline in sperm concentration between 1940 and 1990. Some studies have criticized both the retrospective design and the mathematical analysis used in this study (Brake and Krause, 1992; Bromwich et al., 1994
; Olsen et al., 1995
; Fisch and Goluboff, 1996
).
Some authors (Fisch et al., 1996; Paulsen et al., 1996
; CECOS et al., 1997) suggest that for an accurate assessment of changes in sperm quality, the geographic location must be taken into account. Such geographical differences might be related to ethnic, genetic or environmental factors (Danish Environmental Protection Agency, 1995
; Adamopoulos et al., 1996
; Bujan et al., 1996
).
However, these apparent variations could also be affected by the methods of semen analysis, data processing or population selection (Lipshultz, 1996). An alternative way to investigate possible changes in semen parameters would be to assess by retrospective analysis data from a single laboratory, using the same technique and based on a large population over an extended period of time as the one presented here.
We started this study to investigate whether sperm quality has changed among men in infertile couples referred to our laboratory during the last 36 years (from 1960 to 1996). The purpose was to retrospectively investigate changes in semen parameters (volume, concentration, motility, morphology) of 22 759 men with fertility problems, from North-eastern Spain, living in the greater Barcelona area (rural and urban), and evaluated in a single laboratory by the authors, since 1960.
The possibility of a selection bias due to geographical differences or related to the fact that men in infertile couples do not represent a random sample of the general population, especially because many couples were infertile due to a female factor are recognized. Nevertheless, this population includes the largest number of individuals analysed so far, and the observations over 36 years represent an extended period of time.
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Materials and methods |
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The men were rural and urban people living in the greater Barcelona area from North-eastern Spain who worked in technical or business positions, manufacturing jobs, agriculture and students.
Sample collection
Semen samples were collected by masturbation into wide-mouth glass or plastic containers, supplied by the laboratory, after 37 days of sexual abstinence.
Semen analysis
The following semen parameters were analysed: volume, sperm count, total sperm count, percentage of motile spermatozoa, motile sperm count, percentage of normal spermatozoa and normal sperm count. All analyses were carried out by the authors and the same laboratory technician. Samples were analysed within 3060 min, after liquefaction. The volume was determined by drawing up the entire sample into a 10 ml graduated and contrasted glass tube.
For sperm counts, a Bürker or BürkerTürk haemocytometer (Andolz and Bielsa, 1995) was used with appropriate dilution, depending on the initial impression of sperm concentration (1:10; 1:20; 1:50). Diluting fluid was 10 ml of sodium saline (9 g/l NaCl) or phosphate buffer (0.15 M NaCl) with 100 µl of formaldehyde (35%). Diluted semen samples were mixed before transferring a drop to the two chambers of the haemocytometer. After about 23 min in a moist chamber to allow for the sedimentation of the cells, the spermatozoa were counted under a light microscope at x250 magnification. The number of sperm cells in three of the nine squares on the diagonal of the reticle in each chamber were counted, and the mean value was calculated (World Health Organization, 1992
).
To determine the percentage of motile spermatozoa, a 20 µl drop of gently mixed semen (37°C) was placed on a glass slide (37°C) under a coverslip (20x20 mm). The slide was placed on a heating stage (37°C) and observed at x400 magnification under phase contrast. At least 100 spermatozoa were counted, and the mean value from duplicate measurements was calculated. If the coefficient of variation between the two aliquots was >5%, the determination was repeated in two different aliquots (Andolz and Bielsa, 1995). The percentage of motile spermatozoa was calculated from the ratio of the number of rapidly and slowly moving spermatozoa (grades a and b, according to the WHO classification, 1992) to the total number of spermatozoa counted.
Sperm morphology was assessed in Papanicolaou-stained smears in the first decade (19601969), but in the last period (19701996) all samples were examined using the staining technique, described by us (Bielsa et al., 1994). There was a good correlation between these two methods and ours was easier and provided much more information about the cells. For morphology assessment we used the criteria described by Jöel (1959) and MacLeod (1964) and the classification proposed by David et al. (1975).
Statistical analysis
The data were analysed using SPSS statistical computer software, version 7.5 (SPSS Inc, Chicago, IL, USA). Categorical variables are presented as percentages. All continuous variables are presented as mean ± 1 SD. When their distribution departed from abnormal distribution, median and first and third quartiles are presented. To aid comparison with other authors, e.g. Berman et al. (1996), they are also presented the geometric mean and the 95% confidence interval.
After testing the assumptions on residuals: linearity, equality of variance, independence of error, normality, as well as the examination of the plot of residuals that permitted detection and investigation of outliers, it was concluded that multiple linear regression models were appropriate to assess the effect of age, days of abstinence and calendar year (independent variables) on semen characteristics. Log-transformation (base 10) was required for semen volume, sperm count, total sperm count, percentage of sperm motility, motile sperm count, percentage of normal spermatozoa and normal sperm count. The multiple coefficient of determination (r2) is presented for each model along with P value and the slope of the independent variable of interest which represented the yearly change in percentage. The 2 for trends was used to test whether the percentage of azoospermic men decreased from the 1960s through the 1990s.
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Results |
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Sperm count
There was a non-significant increase (0.04%) over the 36 year period in the spermatozoa group (Figure 1B) (Table III
). The sperm count increased significantly both with increasing age (0.7%; P < 0.004), and increasing period of abstinence (10.0%; P < 0.001). There was a non-significant yearly decline (0.1%) in the total sperm count.
Motile spermatozoa
The percentage sperm motility increased significantly by 0.4% (P < 0.001) in the spermatozoa group (Figure 1C) (Table III
). The percentage of motile spermatozoa decreased significantly with age (0.3%; P < 0.01). In a similar fashion, motility decreased significantly (2.8%; P < 0.001) with increasing abstinence period. There was a non-significant yearly decline (0.2%) in motile sperm count.
Normal spermatozoa
Percentage of normal spermatozoa had a statistically significant decline in our population (3.6%; P < 0.001) (Table III) (Figure 1D
). Abstinence period did not modify the percentage of normal spermatozoa, only age significantly decreased the percentage of normal spermatozoa (0.2%; P <0.001). There was an adjusted 3.4% (r2 = 0.020, P < 0.001) yearly decline in normal sperm count.
Azoospermia
Of the whole population (n = 22 759), 1364 men had azoospermia (6.0%) and 222 (1.0%) were not considered strictly azoospermic because spermatozoa were observed in the pellet after centrifugation. Of the 1364 men with azoospermia, five (0.4%) showed spermatogenic cells in their ejaculate. The percentage of azoospermic men over decades found in our laboratory was as follows: 19601969, 13.6%; 19701979, 6.5%; 19801989, 4.5% and 19901996, 2.5% (P < 0.001 2 for trends). The mean (±SD) age of the azoospermic group was 32.3 ± 5.8) years (range, 1771). The decrease in the percentages with time could be explained because, in more recent times, patients could have been examined elsewhere prior to study in our laboratory.
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Discussion |
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In contrast, our results do not agree with those of other authors who have reported a continuing decrease in sperm count over time (Nelson and Bunge, 1974; Leto and Frensilli, 1981
; Bostofte et al., 1983
; Bendvold, 1989
; Carlsen et al., 1992
; Auger et al., 1995
; Irvine et al., 1996
; Adamopoulos et al., 1996
; Van Waeleghem et al., 1996
).
In the same manner our results do not agree with those of Auger et al. (1995) in a study of 1351 fertile men in Paris, who reported a decrease in the percentage of motile spermatozoa by 0.6%, versus an increase in 0.4% (P < 0.001) in our group. On the other hand, the decrease in the percentage of normal spermatozoa (0.5%) observed by Auger et al. (1995) was lower than those observed in our population (3.6%). These authors showed a significant increase in the age of the sperm donors, from 32 years in 1973 to 36 years in 1992 (P < 0.001). This was not observed in our study (32.4 years in 1960 compared with 33 years in 1996).
Interestingly, the results from our group of 20 411 men are in agreement with those of Berling and Wölner-Hanssen (1997) in infertile men from Southern Sweden, except for the increase in the percentage of normal spermatozoa. Similarly, the results obtained by Bendvold (1989) in infertile Norwegian men are comparable with those of our population (n = 20 411), except for the decrease in semen volume (r2 = 0.006; statistically significant but not clinically important).
None of the changes in semen characteristics observed in this study of 20 411 men indicated deteriorating sperm quality. These changes were probably not clinically significant; the r2 values generally being extremely small. The findings in our population of unselected infertile men agree with those of Fisch et al. (1996) who observed a slight increase in sperm count. Paulsen et al. (1996) also found a slight improvement of sperm concentration in sperm samples from healthy volunteers in Washington state, USA.
The only deteriorating characteristic was the percentage of normal spermatozoa which declined by 3.6% (r2 = 0.248) per year from 1960 to 1996. This parameter varied over time depending on the classification criteria (Joël, 1959; MacLeod, 1964
; David et al., 1975
; World Health Organization, 1987
, 1992
) and evaluation experience. Nevertheless, this was not reflected in the normal sperm count because r2 values were too small (r2 = 0.020).
As it is very difficult to study a large sample of healthy men which represent the general male population, one has to rely on results from large published samples, among which this is the largest published so far. Furthermore, it provides data from a population living in different environmental conditions but in the same region of North-eastern Spain over an extended period of time.
In order to assess the selection bias, a recent report by Handelsman (1997) ilustrates the magnitude of bias due to the use of self-selected volunteers for evaluating sperm output of healthy men in Australia.
Our results do not support the idea that sperm quality is decreasing. However, further prospective and multicentric studies, including representative samples of the general population will be needed to demonstrate whether sperm quality is really decreasing.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 7, 1998; accepted on November 5, 1998.