1 Laboratoire de Biologie de la Reproduction, GREFH, CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme humains), Hôpital Cochin, Université Paris V, Paris, France, 2 Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark, 3 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh, UK and 4 University of Turku, Institute of Biomedicine, Turku, Finland
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Abstract |
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Key words: environmental factors/fertility/spermatogenesis/sperm morphology
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Introduction |
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Several studies suggesting secular and regional variations in human semen quality have recently been reported (Carlsen et al., 1992; Auger et al., 1995
; VanWaeleghem et al., 1996; Fédération CECOS et al., 1997). The possible role of environmental and lifestyle factors in contributing to these variations has been widely discussed, and the studies have sparked off many debates and controversies because their results could be confounded by many factors (Jégou et al., 1999
). There has also been speculation that the observed variations could be due to exposure to environmental chemicals acting as endocrine disrupters (Jensen et al., 1995
; Toppari et al., 1996
). Most of the published data are on sperm concentration, being the semen characteristic most commonly assessed and the one least subject to methodological bias. In contrast, there are fewer studies reporting data on sperm motility and morphology. The assessment of these characteristics is more subjective by nature with an overall noticeable inter-technician and inter-laboratory variability (Neuwinger et al., 1990
; Cooper et al., 1992
; Matson, 1995
; Ombelet et al., 1998
; Auger et al., 2000
). Moreover, sperm motility and morphology assessments are not fully standardized, despite WHO guidelines (WHO, 1992
,1999
), which presents marked difficulties for multicentre studies.
To overcome the problems associated with retrospective studies on semen quality, a prospective multicentre study with a well-standardized protocol has been undertaken in four European cities: Turku (Finland), Copenhagen (Denmark), Edinburgh (UK) and Paris (France). The first results of this study provided clear evidence of geographical variations in sperm concentration and motility (Jørgensen et al., 2001). During this study, microscope slide smears from semen samples collected in each of the four cities were centralized in Paris for Shorr staining and sperm morphology assessment. It has previously been reported that there were no significant geographical difference in normal sperm morphology (Jørgensen et al., 2001
). However, the various profiles of sperm abnormalities allowed an in-depth study of variation in the patterns of sperm abnormalities according to the geographical origin of the men as well as their medical history, environment and lifestyle, for which data were recorded by means of standardized questionnaires.
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Materials and methods |
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Semen samples
All the men were asked to abstain from ejaculation for at least 48 h before semen collection, but were not given any upper limit as we anticipated a reduction in the number of participants if such a limit was imposed upon this group of partners of pregnant women. For each man, a single semen sample was collected by masturbation and ejaculated into a clean collection tube. The assessment of sperm concentration and motility was made according to the then current WHO guidelines (WHO, 1992). Due to the wide inter-laboratory variations in sperm morphology assessment (Neuwinger et al., 1990
; Matson et al., 1995), it was decided to perform this analysis centrally. Semen smears were prepared according to a standardized method in each centre, from a 10 µl drop of the sample, air-dried, then fixed for 1 h with a mixture of absolute ethanol (2/3) and acetic acid (1/3). Each centre sent the unstained coded smears to Paris. The smears were stained using an automatic stainer (Sakura DRS601, Bayer Diagnostics, Puteaux, France) which allows a homogeneity of staining between slides. The staining procedure was Shorr staining according to WHO manuals (WHO, 1992
). For 81 (7.5%) of the 1082 men included in the study the smears could not be assessed, because they did not reach the centre, were broken during transportation or the identification code was not readable. Finally, 1001 slides (Turku = 261, Copenhagen = 294, Edinburgh = 239, Paris = 207) were randomly distributed to five technicians who assessed them blindly. The technicians involved in the study were chosen for their experience and accuracy in sperm morphology assessment as well as their good reproducibility and homogeneity in results, as revealed by regular internal quality controls following WHO recommendations (WHO, 1992
). They had all worked in the laboratory for at least five years, and had on a daily basis assessed five to 10 smears made from semen samples of fertile and infertile men. The regular internal quality controls included the evaluation of intra- and inter-technician variability for the number of morphologically normal sperm and MAI. Just before the beginning of the study, the intra- and inter-individual coefficients of variations for these variables and the five technicians involved were <10%. No marked deviations of their quarterly means for both characteristics were observed.
Method of classification of normal and abnormal spermatozoa
The percentages of morphologically normal spermatozoa and of spermatozoa showing various morphological anomalies were evaluated on 100 sperm at a final x1000 magnification, according to the method described by David et al. and modified after the report of Jouannet et al. ((David et al., 1975; Jouannet et al., 1988
). The modified classification of David et al. distinguishes morphologically normal sperm, seven abnormalities of the head, three abnormalities of the midpiece and five abnormalities of the tail (Figure 1
). Normal sperm and all defects are defined by specific criteria. The originality of David's classification is that all abnormalities observed on each sperm cell are recorded thanks to a multiple-entry system. Thus, no abnormalities are underestimated in relation to another as shown on Figure 1
. The methodology allows the calculation of the MAI (Figure 1
). Other cellular elements (isolated tails, swollen sperm heads, white blood cells, immature germ cells, other cells and cellular debris) are also recorded, but they are not included in the count and they were not analysed in the present study.
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Statistical analysis
All statistics were run using the BMDP statistical software (Dixon, 1988; Statistical Solutions, Cork, Ireland). We first studied the possible confounding effects of the duration of sexual abstinence at collection and age of the men. Since there was no significant relationship between normal sperm morphology or the MAI and sexual abstinence or age, in each city and for all men, these factors were not used as adjustment variables. The equality of means values of the percentages of normal spermatozoa, the percentages of all morphological defects and of MAI between the groups was tested by a one-way analysis of variance (BMDP 7D software) every time there were more than two groups (the possible effect of city, season and alcohol intake) and taking in account cases of unequal variances (Brown-Forsythe test).
When rejecting the null hypothesis, Bonferroni's test, accounting for the variance estimate of all the groups, was used for pairwise mean comparisons.
The equality of means values of the percentages of normal sperm and the percentages of all morphological defects and of MAI when there were only two groups (dichotomic response for a qualitative variable, comparison of two qualitative variables or quantitative variables according to a given threshold) were tested by the distribution-free MannWhitney rank-sum test (BMDP 3D software).
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Results |
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Discussion |
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We found geographical differences for a majority of the sperm defects recorded as well as for the MAI. To our knowledge, this is the first report indicating subtle regional changes in sperm morphogenesis. While such data obviously need to be confirmed by a further investigation in comparable groups of men, it is interesting to note that these differences were found in spite of an absence of differences in the overall proportion of normal sperm. Further investigations are warranted to confirm these findings, which suggest that even if the efficiency of spermiogenesis (in terms of the level of normal sperm release) is geographically stable in comparable healthy fertile men, qualitative differences in the morphogenesis of the head, midpiece and tail exist. We have no meaningful explanation for this phenomenon and the factors modulating these differences are probably numerous and complex.
It could be argued that these results and the other results of the study may be confounded by methodological factors. While we adopted the principle of a centralized assessment, pragmatically, the entire set of slides could not be read by a single technician. We believe this could not have influenced the results of the study since the smears were randomly distributed to the five technicians and blindly assessed, the procedure giving a similar weight to the reader effect in each comparison. Finally, due to the increased variance related to multiple readers, it can be postulated that, at the very worst, existing differences with the factors studied could not be shown for some defects while, on the contrary, the significant differences found, even with a probability level set at 0.10, reflected true differences.
We found significant relationships between some defects and season, but there was no seasonal variation in the percentage of morphologically normal sperm as previously reported in men from infertile couples (Ombelet et al., 1996; Centola and Eberly, 1999
). During the spring compared with the winter, there were less tapered and more microcephalous and multiple heads, shorter and more irregular tails, and fewer cytoplasmic droplets. Overall, we found more tail defects in the spring than in any other season, as recently reported for infertile patients in Rochester, UK (Centola and Eberly, 1999
) while in contrast to the Rochester study, the percentage of cytoplasmic droplets was lowest during the summer. In addition, the mean number of sperm defects per abnormal spermatozoa was higher during the spring than during autumn and winter. While it is known that environmental temperature has an impact on human sperm production (Figa-Talamanca et al., 1992
; Thonneau et al., 1998
), we do not presently know if this can qualitatively affect spermiogenesis and further studies are warranted. It can be speculated that besides the possible impact of the environmental temperature, variations in light exposure and rhythmic changes of lifestyle may act as additional cofactors.
Semen analysis, including sperm morphology assessment, has been suggested to be a useful indicator of the factors in man's macro-environment which can modulate or damage spermatogenesis (MacLeod, 1974; Wyrobek et al., 1983b
). Experimental and occupational studies have shown unambiguously that the mammalian testis is highly vulnerable to numerous physical and chemical factors (Hacker et al., 1981
; Wyrobek et al., 1983a
; Steeno and Pangkahila, 1984
) or more complex factors such as stress (Charpenet et al., 1981
; Fenster et al., 1997
; Yazawa et al., 1999
).
In the present study, we systematically investigated the possible role of a number of antecedent variables on detailed sperm morphology. The answers to the questions on the possible role of factors acting during testicular development and of the urogenital history of the men suggested that some events could have long lasting effects on sperm morphogenesis. The significantly lower percentage of normal spermatozoa and increased percentages of abnormal heads when the mother received treatment during pregnancy evoked the example of diethylstilboestrol (Gill et al., 1979) which was widely used until the 1970s. Maybe other drugs administered during the crucial period of testis development could be implicated. Unfortunately, it was not possible to obtain more details on the type of treatments received.
Concerning the medical history of the man, the most obvious results were related to a history of treatment for one (or both) testi(cle)s not in the scrotum, whatever the anatomical position of the gonad, the nature of the treatment or the age at treatment. Our data suggested that treatments for cryptorchidism, or maybe cryptorchidism by itself, had a major impact on the efficiency of spermiogenesis as indicated by the markedly reduced proportion of normal spermatozoa. In contrast, acquired andrological diseases or iatrogenic factors seemed to have less impact. Notably, we found no effect of a history of Chlamydia infection. Intriguingly, the group of men with lower weights at birth had fewer sperm defects than the other men, when using the median birthweight as a threshold. Similar results were recently reported (Olsen et al., 2000). There is presently no clear explanation of this phenomenon. Using a threshold set at 2500 g reinforced this result, which was not confounded by the duration of gestation (data not shown).
Some factors related to the sociological and lifestyle backgrounds of the men were found to possibly modulate sperm morphogenesis. Among them, perceived stress could have a noticeable impact on sperm morphogenesis for the men reporting daily exposure to mild stress. Similar results were previously reported for healthy volunteers (Giblin et al., 1988) and subfertile men (Bigelow et al., 1998
). It should be pointed out that we found significantly more cytoplasmic droplets and coiled tails in men reporting being exposed to stressful conditions, suggesting that both defects could be putative markers of stressful conditions.
The men who had moderate alcohol habits (3.6 units per week on average) had less sperm defects than those drinking a lot (16.1 units per week on average), as previously suggested (Goverde et al., 1995). Curiously, the men having moderate alcohol consumption had less defects than men who did not drink alcohol at all. We had no biologically meaningful explanation for this intriguing result, which warrants further studies since it is reminiscent of the `French paradox' [the reported low rate of coronary heart diseases related to moderate alcohol consumption (Criqui and Ringel, 1994
)]. There is a body of controversial literature on the effect of smoking on male fertility or semen quality (Ratcliffe et al. 1992
; Vine, 1996
). In the present study, we did not find any strong evidence for a relationship between smoking (smoking versus no smoking or comparisons between various levels of tobacco intake) or passive exposure to smoke during early childhood and sperm defects.
The questions related to occupation referred to broad and heterogeneous occupational categories or to groups of agents (for example, solvents) rather than specific exposures. Although the study was carried out in an unselected population of partners of pregnant womena marked difference to `exposedunexposed' studies including exposure measurementsand only a minority of men was exposed to possibly toxic occupational factors, we observed subtle differences in the number of sperm defects and some occupational `exposures'. However, from the design of the study, methodological flaws may be suspected, but most of the significant differences were consistent with sparse previous literature, for example, on posture at work (Sas and Szöllözi, 1979; Figa-Talamanca et al., 1996
) or metal welding (Bonde, 1992
; Bigelow et al., 1998
). Moreover, these general questions produced intriguing data which provided some trails for further studies; for example, the study indicated that more sperm defects were associated with the lowest rather than the highest weekly working times.
Finally, it should be pointed out that the present data on the relationships found between detailed sperm morphology and the male partners' environment, lifestyle and medical history should not be considered established before further confirmation. Due to the large number of statistical comparisons performed, the possibility cannot be excluded that a number of significant results may have arisen purely by chance. However, a number of these results were expected, based upon previous studies, for example the effect of birthweight, metal welding, treatment during pregnancy and the effect of stress.
In conclusion, the present data indicate that the detailed assessment of the incidence of sperm morphological abnormalities and MAI could be more useful than a simple evaluation of the percentage of normal spermatozoa to study the effect of external factors on human spermatogenesis. The study carried out in a large group of men with recently proven fertility suggests that the `external milieu' may have subtle, complex and sometimes late impacts on the process of human spermiogenesis, and suggests a number of pathways for further exposure studies in humans and/or experimental studies.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 23, 2001; accepted on September 3, 2001.