Sperm morphological defects related to environment, lifestyle and medical history of 1001 male partners of pregnant women from four European cities

J. Auger1,5, F. Eustache1, A.G. Andersen2, D.S. Irvine3, N. Jørgensen2, N.E. Skakkebæk2, J. Suominen4, J. Toppari4, M. Vierula4 and P. Jouannet1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Recently, differences in semen quality have been found among the partners of pregnant women from four European cities: Turku, Copenhagen, Edinburgh and Paris. METHODS: During this study, slides from the four centres were subjected to a centralized assessment of sperm morphology. The percentages of sperm defects were recorded using a multiple-entry classification enabling the calculation of the multiple anomalies index (MAI), which is the mean number of anomalies per abnormal sperm. The relationships between various sperm abnormalities and self-reported data on medical history, lifestyle and occupational factors were examined. RESULTS: Significant differences in the MAI and most of the sperm defects were found between the four cities, and more abnormalities were found in spring than in winter. An increase in some sperm abnormalities was related to medical treatment of the mother during pregnancy, higher birthweight and previous treatment for cryptorchidism. Significant variations of several sperm defects were related to stress, weekly working time, occupational posture and metal welding, suggesting directions for further exposure studies. CONCLUSION: The present study indicated that the detailed assessment of sperm abnormalities is a useful biomarker of the effect of various external factors which may qualitatively affect human spermatogenesis.

Key words: environmental factors/fertility/spermatogenesis/sperm morphology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The morphology of seminal spermatozoa is the end result of a highly complex process of cellular modifications occurring during spermiogenesis. In humans, it leads to widely heterogeneous morphological patterns, with many cellular abnormalities which may be associated with sperm dysfunction. The morphological assessment of human spermatozoa—including the evaluation of the percentage of morphologically normal sperm and the determination of the incidence of various morphological abnormalities—has always been part of semen analysis in couples consulting for infertility [Rowe et al., 1993Go; World Health Organization (WHO), 1999]. It is now well established that the percentage of normal sperm has prognostic value both in vivo (Jouannet et al., 1988Go; Eggert-Kruse et al., 1996Go; Bonde et al., 1998Go) and in vitro (Kruger et al., 1988Go; Toner et al., 1995Go). It has also been reported that the proportion of some specific sperm abnormalities, and the mean number of abnormalities per abnormal spermatozoon [the Multiple Anomalies Index (MAI)] (Jouannet et al., 1988Go; WHO, 1992Go,1999Go) have a prognostic value both in vivo and in vitro (Jeulin et al., 1986Go; Jouannet et al., 1988Go).

Several studies suggesting secular and regional variations in human semen quality have recently been reported (Carlsen et al., 1992Go; Auger et al., 1995Go; 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., 1999Go). There has also been speculation that the observed variations could be due to exposure to environmental chemicals acting as endocrine disrupters (Jensen et al., 1995Go; Toppari et al., 1996Go). 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., 1990Go; Cooper et al., 1992Go; Matson, 1995Go; Ombelet et al., 1998Go; Auger et al., 2000Go). Moreover, sperm motility and morphology assessments are not fully standardized, despite WHO guidelines (WHO, 1992Go,1999Go), 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., 2001Go). 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., 2001Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population
The male partners of pregnant women were approached when they visited the antenatal care units and invited to participate in the study. The inclusion criteria were according to the standardized protocol described by Jørgensen et al. (Jørgensen et al., 2001Go). Briefly, the eligibility criteria for each man were: 20–45 years of age at the time of invitation, living in the local referral area of the hospital to which he was recruited, and born in the country in which he was currently living. Furthermore, the current pregnancy had to be achieved by normal sexual intercourse, and not as a result of any treatment for subfertility or infertility. Participation in the study was accepted even if the man had a past history of urogenital disease or other diseases, as well as any treatment which may affect fertility. Altogether, 1082 men participated in the study; 275 from Turku, 349 from Copenhagen, 251 from Edinburgh and 207 from Paris. The inclusion period in each centre covered at least a full calendar year to take the possible influence of seasonal changes on semen parameters into account.

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, 1992Go). Due to the wide inter-laboratory variations in sperm morphology assessment (Neuwinger et al., 1990Go; 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, 1992Go). 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, 1992Go). 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., 1975Go; Jouannet et al., 1988Go). 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 1Go). 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 1Go. The methodology allows the calculation of the MAI (Figure 1Go). 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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Figure 1. Morphological classification of normal and abnormal human spermatozoa (David et al., 1975Go, modified). The figure provides an illustration of each sperm defect recorded, these sperm defects as well as the morphologically normal spermatozoa being defined by specific criteria (David et al., 1975Go). In this example, 100 spermatozoa have been assessed. Each abnormal spermatozoon with more than one morphological defect is recorded in all the corresponding defect categories, the methodology allowing the calculation of the Multiple Anomalies Index (MAI) (Jouannet et al., 1988Go; WHO, 1992Go and1999Go). {blacklozenge} indicates the categories where an abnormal spermatozoon presenting four morphological defects (thin head, abnormal post-acrosomal region, abnormal or absent acrosome and bent midpiece) is recorded. Note that the total number of isolated and/or associated anomalies is greater than the number of abnormal sperm because all abnormalities of each spermatozoon are recorded in the multiple-entry grid.

 
Variables studied
All the men included in the study completed a standardized questionnaire containing information on previous or current general and urogenital diseases, lifestyle, occupation and on the current pregnancy (Jørgensen et al., 2001Go). The possible influence of 35 recorded variables on detailed sperm morphology was studied. Two variables, city and season, were related to the general environment of the men, 10 variables concerned the medical history of the men (treatment of the mother during pregnancy, birth before term, birthweight, severe disease in the first year of life, treatment because one or both testicles were not in the scrotum, testicular trauma, history of Chlamydia infection, history of prostatitis, surgery for varicocele and surgery for inguinal hernia), seven variables were related to the lifestyle of the subject or his parents [smoking of parents during infancy, age of leaving school, alcohol (beer, wine and spirits) consumption, male and/or female partners smoking, diet exclusively based on organically produced food, satisfaction with sexual life, perception of stressful conditions] and 16 variables concerned the working conditions of the men (weekly working hours, working posture, metal welding, metal turning, metal degreasing, spraying and laying chemicals, house painting, cleaning with organic solvents, photo development, gluing, plastic welding, working with anaesthetics or industrial lacquer or pesticides, working in laboratories, working at temperatures >50°C).

Statistical analysis
All statistics were run using the BMDP statistical software (Dixon, 1988Go; 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 Mann–Whitney rank-sum test (BMDP 3D software).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The mean and median MAI for all combined centres were both equal to 1.58 with 10th and 90th percentiles equal to 1.33 and 1.85 respectively, and the distribution of MAI was normal in each of the four cities. The mean MAI was significantly different among the cities (Table IGo), despite no significant difference in the percentage of normal spermatozoa (Jørgensen et al., 2001Go), with the highest MAI value in Turku and the lowest in Paris. Except for the mean percentages of tapered heads and short tails, there were significant geographical variations in sperm defects with a different geographical pattern according to the category of anomaly assessed (Table IGo). The mean percentage of tail anomalies (the sum of the mean percentages of the five tail anomalies) was significantly higher in Edinburgh than in Paris (P < 0.05), Turku and Copenhagen (both P < 0.01) and there was a significant negative relationship between the percentage of grade a+b WHO motility (WHO, 1992Go) as reported in Jørgensen et al. (Jørgensen et al., 2001Go) and the mean percentage of tail anomalies (r = -0.19, P < 0.0001 by Spearman's rank test, data not shown).


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Table I. Sperm morphology in fertile men from four European cities: geographical variations
 
There were no seasonal variations in normal sperm morphology, while a number of abnormalities and MAI varied significantly according to season (Table IIGo). Most of the significant differences in detailed sperm morphology were found between spring and winter.


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Table II. Sperm morphology in fertile European men: seasonal variations
 
The medical, lifestyle and occupational factors found significantly to modulate the mean percentages of at least two morphological characteristics are presented in Table IIIGo. An increase in some sperm abnormalities was related to medical treatment of the mother during pregnancy, higher birthweight and previous treatment for cryptorchidism. Significant variations of several sperm defects were related to stress, weekly working time, occupational posture and metal welding. Weak variations of the mean percentages of sperm defects were observed according to the following factors. Alcohol consumption: 0 versus 1–6 versus >6 units/week corresponded to a percentage of normal sperm of 48.3, 52.4 and 50.2% (P = 0.017), a mean percentage of cytoplasmic droplets (immature spermatozoa) of 1.3, 1.0 and 0.9 (P = 0.04) and a mean percentage of irregular tails of 0.6, 0.5 and 0.4 respectively (P = 0.029); age of leaving school: 1.3 versus 0.6% cytoplasmic droplets for >=18 years old versus <18 years old (P = 0.0009); metal turning: daily (n = 27) versus never (n = 829) corresponded to a mean percentage of macrocephalous sperm of 0.9 and 0.3 respecively (P = 0.027); and finally, spraying and laying chemicals: daily (n = 36) versus never (n = 814) corresponded to a mean percentage of cytoplasmic droplets of 1.6 and 1.0 respectively (P = 0.033).


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Table III. Sperm morphology in fertile European men: variations related with medical, lifestyle and occupational factors
 
The mean percentage of all sperm anomalies recorded in the fertile men studied was not significantly modulated by the following medical factors: birth before term, severe disease in the first year of life, testicular trauma, a history of Chlamydia infection or prostatitis, and surgery for varicocele or inguinal hernia. It was not modified by the following lifestyle factors: smoking by parents during infancy or by male and/or female partners smoking, diet exclusively based on organically produced food and satisfaction with sexual life. Finally, the mean percentage of all sperm anomalies was not found to be significantly modulated by the following occupational factors: house painting, cleaning with organic solvents, photo development, metal degreasing, gluing, plastic welding, working with anaesthetics, industrial lacquer, pesticide use and working in laboratories and at temperatures >50°C.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study is the first one reporting the overall percentages of sperm defects in a wide population of fertile male partners of pregnant women (Table IGo), providing baseline data for future studies. It should be pointed out that several defects (tapered and macrocephalous heads, cytoplasmic droplets and short tails) were less frequent in the current group of male partners of pregnant women than in other groups of older fertile male candidates for semen donation or vasectomy, who would normally have a long period between the birth of the last child and semen collection (Schwartz et al., 1984Go; Bujan et al., 1988Go).

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., 1996Go; Centola and Eberly, 1999Go). 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, 1999Go) 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., 1992Go; Thonneau et al., 1998Go), 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, 1974Go; Wyrobek et al., 1983bGo). Experimental and occupational studies have shown unambiguously that the mammalian testis is highly vulnerable to numerous physical and chemical factors (Hacker et al., 1981Go; Wyrobek et al., 1983aGo; Steeno and Pangkahila, 1984Go) or more complex factors such as stress (Charpenet et al., 1981Go; Fenster et al., 1997Go; Yazawa et al., 1999Go).

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., 1979Go) 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., 2000Go). 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., 1988Go) and subfertile men (Bigelow et al., 1998Go). 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., 1995Go). 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, 1994Go)]. There is a body of controversial literature on the effect of smoking on male fertility or semen quality (Ratcliffe et al. 1992Go; Vine, 1996Go). 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 women—a marked difference to `exposed–unexposed' studies including exposure measurements—and 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, 1979Go; Figa-Talamanca et al., 1996Go) or metal welding (Bonde, 1992Go; Bigelow et al., 1998Go). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Cynthia LeBon, Francioise Roques, Marie Rossi, Sidi El Matribi and Gérard Limea for their excellent technical assistance in sperm morphology assessment and Catherine Pauzat and Jean Claude Juillard for data collection. The study was supported by contract BMH4-CT96-0314 from the European Union, a French research grant (1752) from Direction des Recherches, Etudes, et Technologies, Ministère de l'Education Nationale and the Finnish Research Programme on Environmental Health, Academy of Finland.


    Notes
 
5 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Hôpital Cochin, 123 Bd de Port-Royal, 75014 Paris, France. E-mail: jacques.auger{at}cch.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Auger, J., Kunstmann, J.M., Czyglik, F. and Jouannet, P. (1995) Decline in semen quality among fertile men in Paris during the last 20 years. New Engl. J. Med., 332, 281–285.[Abstract/Free Full Text]

Auger, J., Eustache, F., Ducot, B. et al. (2000) Intra- and inter-individual variability in human sperm concentration motility and vitality assessment during a workshop involving 10 laboratories. Hum. Reprod., 15, 2360–2368.[Abstract/Free Full Text]

Bigelow, P.L., Jarrell, J., Young, M.R. et al. (1998) Association of semen quality and occupational factors: comparison of case–control analysis and analysis of continuous variables. Fertil. Steril., 69, 11–18.[ISI][Medline]

Bonde, J.P. (1992) Semen quality in welders exposed to radiant heat. Br. J. Ind. Med., 49, 5–10.[ISI][Medline]

Bonde, J.P., Ernst, E., Jensen, T.K. et al. (1998) Relation between semen quality and fertility: a population-based study of 430 first-pregnancy planners. Lancet, 352, 1172–1177.[ISI][Medline]

Bujan, L., Mieusset, R., Mondinat, C. et al. (1988) Sperm morphology in fertile men and its age related variation. Andrologia, 20, 121–128.[ISI][Medline]

Carlsen, E., Giwercman, A., Keiding, N. and Skakkebaek, N.E. (1992) Evidence for decreasing quality of semen during past 50 years. Br. Med. J., 305, 609–613.[ISI][Medline]

Centola, G.M. and Eberly, S. (1999) Seasonal variations and age-related changes in human sperm count, motility, motion parameters, morphology, and white blood cell concentration. Fertil. Steril., 72, 803–808.[ISI][Medline]

Charpenet, G., Tache, Y., Forest, M.G. et al. (1981) Effects of chronic intermittent immobilization stress on rat testicular androgenic function. Endocrinology, 109, 1254–1258.[Abstract]

Cooper, T.G., Neuwinger, J., Bahrs, S. and Nieschlag, E. (1992) Internal quality control of semen analysis. Fertil. Steril., 58, 172–178.[ISI][Medline]

Criqui, M.H. and Ringel, B.L. (1994) Does diet or alcohol explain the French paradox? Lancet, 344, 1719–1723.[ISI][Medline]

David, G., Bisson, J.P., Czyglik, F. et al. (1975) Anomalies morphologiques du spermatozoïde humain. 1) Propositions pour un système de classification. J. Gynecol. Obstet. Biol. Reprod., suppl. 1,17–36.

Dixon, W.J. (1988) BMDP Statistical Software Manual. University of California Press, Berkeley.

Eggert-Kruse, W., Schwarz, H., Rohr, G. et al. (1996) Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception. Hum. Reprod., 11, 139–146.[Abstract]

Fédération CECOS, Auger, J. and Jouannet, P. (1997) Evidence for regional differences of semen quality among fertile french men. Hum. Reprod., 12, 740–745.[Abstract]

Fenster, L., Katz, D.F., Wyrobek, A.J. et al. (1997) Effects of psychological stress on human semen quality. J. Androl., 18, 194–202.[Abstract/Free Full Text]

Figa-Talamanca, I., Dell'Orco, V., Pupi, A. et al. (1992) Fertility and semen quality of workers exposed to high temperatures in the ceramics industry. Reprod. Toxicol., 6, 517–523.[ISI][Medline]

Figa-Talamanca, I., Cini, C., Varricchio, G.C. et al. (1996) Effects of prolonged autovehicle driving on male reproduction function: a study among taxi drivers. Am. J. Ind. Med., 30, 750–758.[ISI][Medline]

Giblin, P.T., Poland, M.L., Moghissi, K.S. et al. (1988) Effects of stress and characteristic adaptability on semen quality in healthy men. Fertil. Steril., 49, 127–132.[ISI][Medline]

Gill, W.B., Schumacher, G.F., Bibbo, M. et al. (1979) Association of diethylstilbestrol exposure in utero with cryptorchidism, testicular hypoplasia and semen abnormalities. J. Urol., 122, 36–39.[ISI][Medline]

Goverde, H.J., Dekker, H.S., Janssen, H.J. et al. (1995) Semen quality and frequency of smoking and alcohol consumption—an explorative study. Int. J. Fertil. Menopausal. Stud., 40, 135–138.[Medline]

Hacker, U., Schumann, J., Gohde, W. and Muller, K. (1981) Mammalian spermatogenesis as a biologic dosimeter for radiation. Acta. Radiol. Oncol., 20, 279–282.[ISI][Medline]

Jégou, B., Auger, J., Multigner, L. et al. (1999) The saga of sperm count decrease in humans and wild and farm animals. In Gagnon, C. (ed.) The Male Gamete: From Basic Knowledge to Clinical Applications. Cache River Press, Vienna, pp 445–454.

Jensen, T.K., Toppari, J., Keiding, N. and Skakkebaek, N.E. (1995) Do environmental estrogens contribute to the decline in male reproductive health? Clin. Chem., 41, 1896–1901.[Abstract/Free Full Text]

Jeulin, C., Feneux, D., Serres, C. et al. (1986) Sperm factors related to failure of human in-vitro fertilization. J. Reprod. Fertil., 76, 735–744.[Abstract]

Jørgensen, N., Andersen, A.G., Eustache, F. et al. (2001) Regional differences in semen quality in Europe. Hum. Reprod., 16, 1012–1019.[Abstract/Free Full Text]

Jouannet, P., Ducot, B., Feneux, D. and Spira, A. (1988) Male factors and the likelihood of pregnancy in infertile couples. I. Study of sperm characteristics. Int. J. Androl., 11, 379–384.[ISI][Medline]

Kruger, T.F., Acosta, A.A., Simmons, K.F. et al. (1988) Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil. Steril., 49, 112–117.[ISI][Medline]

MacLeod, J. (1974) Effects of environmental factors and of antispermatogenic compounds on the human testis as reflected in seminal cytology. In Mancini, R.E and Martini, L. (eds) Male Fertility and Sterility. Academic Press, New York, pp 123–148.

Matson, P.L. (1995) External quality assessment for semen analysis and sperm antibody detection: results of a pilot scheme. Hum. Reprod., 10, 620–625.[Abstract]

Neuwinger, J., Behre, H.M. and Nieschlag, E. (1990) External quality control in the andrology laboratory: an experimental multicenter trial. Fertil. Steril., 54, 308–314.[ISI][Medline]

Olsen, J., Bonde, J.P., Basso, O. et al. (2000) Birthweight and semen characteristics. Int. J. Androl., 23, 230–235.[ISI][Medline]

Ombelet, W., Maes, M., Vandeput, H. et al. (1996) Chronobiological fluctuations in semen parameters with a constant abstinence period. Arch. Androl., 37, 91–96.[ISI][Medline]

Ombelet, W., Bosmans, E., Janssen, M. et al. (1998) Multicenter study on reproducibility of sperm morphology assessments. Arch. Androl., 41, 103–114.[ISI][Medline]

Ratcliffe, J.M., Gladen, B.C., Wilcox, A.J. and Herbst, A.L. (1992) Does early exposure to maternal smoking affect future fertility in adult males? Reprod. Toxicol., 6, 297–307.[ISI][Medline]

Rowe, P.J., Comhaire, F.H., Hargreave, T.B. and Mellows, H.J. (1993) WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge University Press, Cambridge, pp. 33–39.

Sas, M. and Szöllözi, J. (1979) Impaired spermiogenesis as a common finding among professional drivers. Arch. Androl., 3, 57–60.[ISI][Medline]

Schwartz, D., Mayaux, M.J., Guihard-Moscato, M.L. et al. (1984) Study of sperm morphologic characteristics in a group of 833 fertile men. Andrologia, 16, 423–428.[ISI][Medline]

Steeno, O.P. and Pangkahila, A. (1984) Occupational influences on male fertility and sexuality. Andrologia, 16, 5–22.[ISI][Medline]

Thonneau, P., Bujan, L., Multigner, L. and Mieusset, R. (1998) Occupational heat exposure and male fertility: a review. Hum. Reprod., 13, 2122–2125.[Abstract]

Toner, J.P., Mossad, H., Grow, D.R. et al. (1995) Value of sperm morphology assessed by strict criteria for prediction of the outcome of artificial (intrauterine) insemination. Andrologia, 27, 143–148.[ISI][Medline]

Toppari, J., Larsen, J.C., Christiansen, P. et al. (1996) Male reproductive health and environmental xenoestrogens. Environ. Health Perspect., 104, Suppl. 4,741–803.[ISI][Medline]

Van Waeleghem, K., De Clercq, N., Vermeulen, F. et al. (1996) Deterioration of sperm quality in young healthy Belgian men. Hum. Reprod., 11, 325–329.[Abstract]

Vine, M.F. (1996) Smoking and male reproduction: a review. Int. J. Androl., 19, 323–337.[ISI][Medline]

WHO (1992) World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.

WHO (1999) World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.

Wyrobek, A.J., Gordon, L.A., Burkhart, J.G. et al. (1983a) An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman animals: a report of the US Environmental Protection Agency Genetox program. Mutation Res., 115, 1–72.[ISI][Medline]

Wyrobek, A.J., Gordon, L.A., Burkhart, J.G. et al. (1983b) An evaluation of human sperm as indicators of chemically induced alterations of spermatogenesis in man. Mutation Res., 115, 73–148.[ISI][Medline]

Yazawa, H., Sasagawa, I., Ishigooka, M. and Nakada, T. (1999) Effect of immobilization stress on testicular germ cell apoptosis in rats. Hum. Reprod., 14, 1806–1810.[Abstract/Free Full Text]

Submitted on May 23, 2001; accepted on September 3, 2001.