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Air-conditioned environments do not prevent deterioration of human semen quality during the summer*
FERTILITY AND STERILITY
Vol. 57, No.5, May 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Air-conditioned environments do not prevent deterioration of human semen quality during the summer*
Richard J. Levine, M.D.t:j: Michelle H. Brown, B.S.P.H.t Michelle Bell, M.S.§ II
Frances Shue, M.D.§ Gary N. Greenberg, M.D.~ Brenda L. Bordson, Ph.D.§
Chemical Industry Institute of Toxicology, Research Triangle Park; Duke University Medical Center, Durham, North Carolina; Reproductive Resources Inc., Metairie; and Tulane University, New Orleans, Louisiana
Objective: To determine if air conditioning might mitigate summer reductions in semen quality. Design: Prospective study of semen quality in summer and winter. Setting: Normal human volunteers were studied in the setting of a fertility laboratory. Patients, Participants: Two groups of volunteers were selected from the vicinity of New Orleans: 64 men who worked indoors during the summer in air-conditioned environments and 76 others who worked outdoors. Interventions: None. Main Outcome Measures: Parameters of manual semen analysis were examined for seasonal and group differences. Results: Remarkably similar reductions in semen quality during summer as compared with winter were observed in both indoor and outdoor workers, respectively, with regard to the following parameters of semen quality: 19% and 19% in sperm concentration, 25% and 27% in total sperm per ejaculate, 17% and 20% in motile sperm concentration, 13% and 15% in percent sperm with normal morphology, and 23% and 23% in concentration of morphologically normal motile sperm. Conclusions: These findings do not support the hypothesis that the heat of the summer is detrimental to male reproductive capacity. The available evidence suggests instead a possible role of photoperiod in causing the seasonal changes in semen quality. Fertil Steril 1992;57:1075-83 Key Words: Semen, sperm, summer, winter, heat, photoperiod, air-conditioning
Human sperm concentration, total sperm count per ejaculate, and motile sperm concentration are
Received March 15, 1991; revised and accepted January 8, 1992. * Supported by the Chemical Industry Institute of Toxicology; Duke University Medical Center, Division of Occupational and Environmental Medicine; and Reproductive Resources Inc. t Chemical Industry Institute of Toxicology. t Reprint requests and present address: Richard J. Levine, M.D., Epidemiology Branch; Division of Epidemiology, Statistics, and Prevention Research; National Institute of Child Health and Human Development; National Institutes of Health, EPN 640, Bethesda, Maryland 20892. § Reproductive Resources Inc. II Department of Cellular and Molecular Biology, Tulane University. ~ Division of Occupational and Environmental Medicine, Duke University Medical Center. Vol. 57, No.5, May 1992
reduced during the summer season (1, 2). Could the heat of the summer cause the quality of semen to deteriorate? If so, men who work outdoors in a warm climate during the middle of the day should exhibit a greater decline in sperm concentration than those who work indoors in air-conditioned environments. We report here the results of a prospective study of semen quality during July to August 1989 and January to February 1990 among indoor and outdoor workers in New Orleans (average maximal daily temperature 31.9°C in June and July 1989 and 17.0°C in December 1989 and January 1990) (3). MATERIALS AND METHODS Subjects
Men who worked indoors or outdoors during the summer were recruited in the summer of 1989 Levine et al.
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through newspaper advertisements and asked to visit a reproductive laboratory once that summer and once the following winter. At each visit the men were interviewed, and they provided fresh semen specimens. Summer visits took place within the period July 15 to August 16, 1989 (median, July 31 for indoor workers, July 27 for outdoor workers); winter visits occurred within the period January 12 to February 16, 1990 (median, January 26 for both indoor and outdoor workers). All men gave informed consent for the study and were paid for their participation. Outdoor workers were defined as persons who worked outdoors during summer at least 4 hours per day between 8:00 A.M. and 6:00 P.M. All other dayshift workers were considered to be indoor workers except persons who worked indoors in warm environments (e.g., cook, baker, lifeguard at an indoor swimming pool), who were excluded from the study. Of 224 volunteers who visited the laboratory during the summer, 178 (76 indoor, 102 outdoor workers) were asked to return the next winter. The others were discarded for the following reasons: in collecting semen specimens 19 reported a loss of seminal fluid exceeding a drop; 12 were judged to be unreliable informants or of an undesirable character; 9 could not be classified as either indoor or outdoor workers; and 6 did not produce suitable semen specimens. A total of 142 men (65 indoor, 77 outdoor workers), or 80% of those requested, did in fact return during the winter. Satisfactory winter specimens were obtained from all of these, but 3 men reported a collection loss greater than a drop and were required to provide a substitute specimen a week later. Questionnaires
At each laboratory visit the subjects were asked how long it had been since their most recent ejaculation «24 hours or a specific number of days), whether during the previous 3 months they had had any febrile illness or used any medications (and if so, the medications used), and whether they had collected their specimens completely. Information was obtained about the type of work performed, the duration of employment, the usual time for starting and finishing work, the number of hours per day at work spent outdoors or in air-conditioned settings within the past 3 months, and the number of hours per day spent outdoors when not at work. Subjects were also questioned about the presence of any urethral discharge or dysuria within the past month or any testicular pain within the past 3 months, con1076
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sumption of alcoholic beverages during the past 3 months, and cigarette smoking history including the number of cigarettes smoked per day during the past 3 months. Laboratory Methods
Before semen collection, subjects were requested to abstain from ejaculation for at least 2 days. Fresh ejaculates were produced at the laboratory by masturbation into sterile plastic containers. Specimens were allowed to liquefy at room temperature. If liquefaction was incomplete, semen analysis was begun 45 minutes after specimen collection. Laboratory analysis of semen volume and sperm count and motility was performed by one laboratory technician. This technician also made an initial assessment of sperm morphology from stained slides shortly after each specimen had been obtained. When all specimens had been collected, identifying information was concealed, and slides were thoroughly shuffled. Then a second, more experienced observer, "blinded" with regard to season and subject, read the slides for sperm morphology. Only the quantitative data on sperm morphology obtained by the blinded, more experienced observer is reported here. Later another experienced observer, also blinded, read the slides again for sperm morphology. After the determination of seminal fluid volume with a graduated syringe, the concentration of spermatozoa was measured with a Neubauer hemocytometer (Reichert-Jung Leica Inc., Buffalo, NY), using a 1:20 dilution of semen to diluent. To arrive at sperm concentration of X106/mL, sperm with tails were counted in 5 of the 25 large squares in the central portion of the hemocytometer. Those on the edge of a square were included in the count if the greater portion of the sperm head fell within the square. Total sperm per ejaculate was the product of sperm concentration and semen volume. For motility assessment, a wet preparation was made by placing a 15-ttL drop of undiluted semen between a slide and a coverslip. A total of 100 sperm (or 4 to 6 microscopic fields) was scanned using a phase-contrast microscope. The motility of each sperm was classified as follows: 0, immotile; 1, nonprogressively motile; 2, weak progression; 3, moderate progression; 4, excellent progression. Percent motile sperm was computed as the percentage of sperm with motility grades 1 to 4. The motility grade of the motile sperm in a specimen was determined by multiplying the number of sperm in each category of motile sperm by the score assigned to that cateFertility and Sterility
gory (1, 2, 3, or 4) and dividing by the total number of motile sperm. To evaluate sperm morphology, a drop of fresh semen was smeared on a slide, air dried, fixed with ethanol, and stained using Gram crystal violet and Gram safranin. One hundred sperm were assigned to the following categories: small head, large head, two heads, two tails, tapered head, coiled tail, bent midpiece, cytoplasmic droplet present, pyriform head, amorphous head, or normal (i.e., absence of the foregoing characteristics). Statistical Analysis
Number of Subjects and Specimens
Of the 142 subjects who provided specimens in both summer and winter, 2 (1 indoor, 1 outdoor worker) who were azoospermic in both seasons were excluded because seasonal variation in semen quality cannot be detected in men without sperm. Three indoor workers who had been laboratory sperm donors before the study and were known to be highly fertile were removed from the comparisons of indoor and outdoor workers. The participation of these subjects had been solicited and was not in response to the newspaper advertisements. Had they been included in the between-group comparisons, aggregate values of the indoor workers would have been elevated unfairly. These three men, however, were retained for the within-person comparisons of semen quality in summer and winter. Because of the likelihood of laboratory error, one indoor worker with an extreme summer-winter concentration difference (summer ~ winter) was omitted from analyses of concentration-related measures. In another indoor worker for whom winter motility data were missing, parameters of sperm motility could not be compared between summer and winter. Slides prepared for sperm morphology had not been retained for the summer specimens of 13 study participants (3 indoor workers, 10 outdoor workers). The winter slide of another subject contained too few sperm to evaluate. After removing these 14 subjects, a total of 126 men with morphological analyses in both summer and winter was used to assess the effect of season on sperm morphology. All 260 specimens remaining after excluding the 3 indoor workers who had been laboratory sperm donors were used in the within-season comparisons of indoor and outdoor workers. The winter specimens of the 13 subjects without summer slides and the summer specimen of the man whose winter slide could not be evaluated were included in these comparisons. Vol. 57, No.5, May 1992
Significance of Paired Seasonal Differences
For comparisons across seasons within subjects, paired t-tests and ANOVA were employed to assess the effect of season on abstinence and on semen quality wherever distributions could be normalized. Arithmetic transformations were needed for all variables except counts of morphologically normal sperm. Cube-root transformations were used for ejaculate volume, sperm concentration, total sperm count per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm. A log transformation was used for abstinence. The distributions of the frequencies of abnormal morphological types could not be normalized by cube root, square root, or log transformations. These variables were, therefore, analyzed with Wilcoxon's signed rank test. This nonparametric method was also applied to the untransformed variables mentioned above. Because it led to the same conclusions as the parametric tests on the normalized data, only results of the latter are reported. Significance of Differences Between Indoor and Outdoor Workers
T-tests were used to determine if arithmetic summer-winter differences in concentration-related parameters of semen quality (sperm concentration, total sperm count, motile sperm concentration, and concentration of morphologically normal motilesperm) varied significantly between indoor and outdoor workers. Analyses of variance were employed to assess the significance of within-season differences in concentration-related variables. Nonparametric analyses of within-season differences between groups were also performed on the untransformed variables, with the Kruskal-Wallis and the van der Waerden (normal scores) tests. Because these yielded the same conclusions as the parametric tests, only the results of the latter are reported. Statistical power calculations were performed using standard methods (4, 5). Control of Confounders
To control for potential confounding characteristics, the ANOV As were enhanced by the addition of measures of possible confounding. The following continuous variables were added as covariates: abstinence (specific number of days, or a half day if <24 hours) in the inverse scale, current smoking (packs of cigarettes per day), and current drinking (drinks per month). Fever and symptoms of possible urogenital disease (urethral discharge, dysuria, and Levine et al.
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Table 1
Characteristics of Seminal Fluid Samples Obtained From 64 Indoor Workers in Summer and Winter Summertime change *
Unadjusted values
Unadjusted %:j:
Adjusted %:j:§
2.5 72.2 171.3 45.1
-2 -18 -25 -14
(-12, 10) (-32, 7) (-38,9) (-23, -5)g
-2 -19 -25 -13
52.6 2.9 41.1 19.8
-2 -1 -15 -22
(-8, 4) (-7,6) (-35,6) (-46,4)'
0(-7,6) 0(-6,7) -17 (-35,13) -23 (-45, 2)"
Variablet
Winter
Summer
Volume of ejaculate (mL) I Sperm concentration (X10 6 jmL) I a Total sperm per ejaculate (X10 6 ) II" Sperm with normal morphology (%)C Motile sperm Percent" Motility grade" Concentration (X10 6 jmL) lib Concentration with normal morphology (X10 6 jmL) lid
2.5 88.3 229.4 52.4 53.7 3.0 48.4 25.5
(-11, (-31, (-36, (-21,
10) 12) 13) -5)g
* Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "63 subjects, b62 subjects, C61 subjects, and d 59 subjects. :j: Two-tailed test for the significance of the seasonal difference; "P < 0.10, 'p < 0.05, and gp < 0.005.
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. I A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
testicular pain) were added as binary variables according to season-specific yes-no categories. Collection losses could not confound the results because none of the specimens used for analysis had collection losses in excess of a drop. Medications were not a problem because no subjects reported use of pharmaceuticals known to affect semen quality, such as anticancer, nitrofuran, or sulfa drugs.
During the summer of 1989, the subjects who were outdoor workers spent an average of 7.5 hid at work outdoors (6.5 hid during winter) and 0.5 hours in air-conditioned surroundings. These circumstances were reversed among indoor workers, who spent 0.5 hid outdoors and 8.5 hid in air-conditioned surroundings. Even when not at work, outdoor workers spent more daytime outdoors than indoor workers (in summer 1 hour versus 0.5 hid during the week, 3.5 hours versus 2 hid on the weekend). Indoor workers were employed largely in professional, technical, and managerial occupations (55%) or in clerical and sales occupations (19%). The most prevalent job categories among outdoor workers were structural work occupations (42%, mostly construction trades, such as welder, electrician, painter, carpenter, brick mason, roofer), agricultural occupations (18%), and miscellaneous occupations (22%) including driving a truck and servicing vehicles (8). The seasonal characteristics of the seminal-fluid samples from the 64 indoor workers, the 76 outdoor workers, and all 140 subjects without azoospermia are presented in Tables 1,2, and 3. The sperm concentration, total sperm count per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm among individual men were lower in summer than in winter. Many of the unadjusted reductions were statistically significant. Reductions for indoor workers (18%, 25%, 15%, and 22%, respectively) were somewhat less than those for outdoor workers (24%,33%,23%, and 25%, respectively), but disparities between the groups with respect to the summer-winter difference for any
Confidence Limits
Two-sided 95% confidence limits (Cl) were determined for the difference between summer and winter for each measure of semen quality, with SEs from the ANOVA (6, 7). Confidence limits were then expressed as a percentage of the winter's mean value. For the analyses conducted in the cube-root scale, the confidence limits are reported after retransformation to the natural scale. RESULTS
Of the 140 subjects without azoospermia who were used in the analysis, 102 were white, 37 were black, and 1 was mixed race of French descent (Creole). The proportion of nonwhite men was similar among indoor and outdoor workers (28% and 26%, respectively). The men ranged from 18 to 57 years of age (mean of 30 for both indoor and outdoor workers). Outdoor workers were more likely than indoor workers to be current smokers (64% versus 36%, respectively) and to consume two or more drinks per day of beer, wine, or spirits (39% versus 13%). 1078
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Fertility and Sterility
Table 2
Characteristics of Seminal Fluid Samples Obtained From 76 Outdoor Workers in Summer and Winter Summertime change'
Unadjusted values Variablet
Winter
Summer
Volume of ejaculate (mL) II Sperm concentration (X10 6 jmL) II Total sperm per ejaculate (X10 6 ) I Sperm with normal morphology (%)" Motile sperm Percent Motility grade Concentration (X10 6 jmL) II Concentration with normal morphology (X10 6 jmL) II"
2.3 73.4 174.5 50.9
2.3 55.8 116.6 43.8
52.8 2.9 41.6 22.3
52.9 2.8 32.1 16.7
Unadjusted %:j:
Adjusted %:j:§
-2 -24 -33 -14
3 -19 -27 -15
(-10, (-34, (-40, (-24,
10) -2)C O)C -4)d
0(-7,7) -3 (-9,4) -23 (-36, l)b -25 (-47, -6)C
(-7, 18) (-30, 10) (-34, 19) (-28, -3)C
0(-8,8) -1 (-8, 7) -20 (-33, 13) -23 (-45,9)
• Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "65 subjects. :j: Two-tailed test for the significance of the seasonal difference; bp < 0.10, cp < 0.05, and dp < 0.005.
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. I A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
measure of semen quality did not achieve significance. There were no significant differences between indoor and outdoor workers in regard to the measures of summer semen quality itself. Morphologically normal sperm decreased in each group by 14% during summer as compared with winter, a change that was highly statistically significant. There were minimal differences in the length of the abstinence period preceding specimen collection between indoor and outdoor workers and between summer and winter seasons. In the combined groups, the values for sperm concentration obtained in winter exceeded the sum-
mer values in 59% of the subjects. However, the proportion of men with sperm concentrations < 20 X 106/mL was lower in summer than in winter (20 of 139 or 14% in summer versus 26 of 139 or 19% in winter), a difference that was not statistically significant. Adjustments were made with linear models for the confounding influence of length of abstinence, cigarette smoking, consumption of alcoholic beverages, symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain), and fever, considered jointly. The magnitude of the summer reduction in sperm concentration, total
Table 3
Characteristics of Seminal Fluid Samples Obtained From 64 Indoor and 76 Outdoor Workers in Summer and Winter Unadjusted values
Summertime change*
Variablet
Winter
Summer
Unadjusted %:j:
Volume of ejaculate (mL) I Sperm concentration (X10 6 jmL) II" Total sperm per ejaculate (X10 6 ) II" Sperm with normal morphology (%)C Motile sperm Percent" Motility grade" Concentration (X10 6 jmL) lib Concentration with normal morphology (X10 6 jmL) lid
2.4 80.1 199.3 51.7
2.4 63.2 141.4 44.4
-2 (-8,7) -21 (-28, -4)f -29 (-34,-W -14 (-20, -8)h
1 -20 -26 -15
53.2 2.9 44.7
52.7 2.9 36.1
-1 (-6,4) -2 (-6,3) -19 (-30, -3)f
-1 (-5,4) 0(-5,4) -19 (-28, 3)·
23.9
18.2
-24 (-41, -12)"
-24 (-40, -7) f
* Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "139 subjects, b138 subjects, c126 subjects, and d124 subjects. :j: Two-tailed test for the significance of the seasonal difference; .p < 0.10, fp < 0.02, .p < 0.001, and hp < 0.0001. Vol. 57, No.5, May 1992
Adjusted %:j:§ (-6, 10) (-26, 1)· (-28, 5) (-22, -8)h
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. II A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
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sperm per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm was altered little after such adjustments (Tables 1, 2, and 3), but differences in the extent of reduction between indoor and outdoor workers narrowed (19%, 25%, 17%, and 23% among indoor workers versus 19%, 27%, 20%, and 23% among outdoor workers, respectively). After adjustment, the concentration of morphologically normal motile sperm continued to be significantly reduced during summer among all subjects, and the percentage of morphologically normal cells remained significantly lower in summer in both groups of workers. Besides season and abstinence, no variable explained > 1 % of the conditional within -subject variance of the concentration-related measures and the percentage of morphologically normal cells. The power of this study to detect any but a large difference between indoor and outdoor workers in sperm concentration was small. The true summer sperm concentration among outdoor workers must be at least 41 % less than that of indoor workers for there to have been 80% likelihood of observing a significant difference; a much larger percent increase in the summer-winter difference in sperm concentration is needed among outdoor workers for their seasonal variation to have been as likely to exceed that of indoor workers significantly. Analyses of variance with or without confounding variables revealed no significant correlations between the number of hours per day spent outdoors at work during the summer and the summer values of concentration-related measures of semen quality and the percentage of sperm with normal morphology. There were no significant correlations with the differences between summer and winter values. During the initial unblinded assessment of sperm morphology, the percentage of sperm with normal morphological features was found to be significantly greater during the summer. A winter increase in the frequency of sperm with cytoplasmic droplets accounted for most of the difference. A significant summer increase in the frequency of sperm with tapered heads was also observed. After the completion of specimen collection when all slides were read by the first experienced, blinded observer, a highly significant decrease in the proportion of sperm with normal morphology was found in the summer. This resulted from significant (or near significant) summer increases in the proportion of sperm with ta~ pered or pyriform heads, detected in both indoor and outdoor workers. Sperm with cytoplasmic droplets were found to be slightly more frequent during 1080
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the summer. The second experienced, blinded observer also observed a highly significant (P < 0.004) decrease in the proportion of sperm with normal morphology during summer. This observer found significant summer increases in sperm with small, tapered, or amorphous heads and in sperm with cytoplasmic droplets. DISCUSSION
We conducted pairwise analyses of semen specimens obtained in summer and winter from men who worked indoors in air-conditioned environments or outdoors during the summer in the vicinity of New Orleans. Substantial summer reductions of sperm concentration, total sperm per ejaculate, motile sperm concentration, percent sperm with normal morphology, and concentration of morphologically normal motile sperm were revealed among both groups of workers. After adjustment for potential confounding factors, seasonal differences remained substantial but were statistically significant within each group only for percent sperm with normal morphology. Because adjusted summer-winter differences were similar in indoor and outdoor workers, the groups could logically be combined to provide a more powerful test of the overall seasonal effect in New Orleans workers. After adjustment, summer reductions in most concentration-related measures of semen quality and in the percentage of sperm with normal morphology were significant or of borderline significance in the combined groups. The results confirm the deterioration in sperm concentration and related parameters of semen quality that has been noted repeatedly during the summer at various nonequatorial locations in the northern hemisphere (1). Moreover, they support the finding of a summer reduction in the percentage of sperm with normal morphological features, previously reported in a retrospective study of patients at a New Orleans fertility clinic (9). In that study, among 1,159 samples contributed by 903 men, there were significantly fewer sperm with normal morphological features during summer than in fall, winter' and spring. However, this was not observed in pairwise comparisons among the 61 men who had provided specimens in both summer and other seasons. A significant summer excess of sperm with bent midpieces was detected among all samples, whereas in the present study, sperm with tapered or pyriform heads were found to be significantly increased. Differences between morphology assessments made by the first technician and the others were Fertility and Sterility
striking. The first technician was less experienced, was aware of the season in which the specimens had been obtained, and analyzed slides in the order in which they had been made, with a gap of 6 months separating analyses of the summer and winter samples. It may have been difficult, therefore, for this technician to maintain consistency across seasons in the subjective procedure of reading slides. This could have resulted in a more critical approach taken toward the winter specimens. Besides being more experienced, the second and third technicians were blinded as to season as well as subject and completed all analyses in random order within 2 to 3 weeks. All three technicians reported a significant excess of tapered sperm heads during the summer season. Observer bias across seasons may have influenced the measurements of sperm motility and progression. Because these analyses require fresh semen, it was not possible to blind the laboratory technician with respect to season. Despite the potential for bias, no seasonal difference was observed in the percentage of motile sperm or in motility grade. A similar study in San Antonio in which automated semen analyses had been performed with a computerized image analysis system, thus reducing the likelihood for bias, found no summer-winter differences in the percentage of motile sperm or in the characteristics of sperm movement (velocity, linearity, linear velocity, mean amplitude of lateral head displacement, and beat/cross frequency) (2). Values for semen volume and sperm concentration, which were determined from fresh semen through more objective means, are less likely to be biased. In assessing sperm concentration, the component of the measurement process most susceptible to observer inconsistency would appear to be judging whether sperm with heads on the edge of a square should be included in the count. However, even if all sperm on the edges of the squares were included for the winter specimens (equivalent to enlarging each of the squares' two dimensions by the length of a sperm head) and none for the summer, the resulting difference can be calculated to be <5%, far less than the 21 % actually observed. A summertime increase in the proportion of men with sperm concentrations < 20 X 106/mL (oligospermia) and considered at higher risk of infertility (10) was not detected in this study. The similar study of 131 men in San Antonio had found oligospermia to be considerably more frequent during the summer (2). Although the two studies involved comparable numbers of subjects, in the latter study sperm concentrations in each season were usually determined Vol. 57, No.5, May 1992
from the mean of two specimens. Seasonal values for sperm concentration, therefore, were more stable, enhancing the potential for detecting nonrandom change. Besides increasing the risk of infertility and the chance that pregnancy will not occur, deterioration of semen quality in a population may affect fertility rates by lengthening the time to pregnancy (11). It is possible that the summer reduction in semen quality may contribute to the deficit in spring births that occurs in nonequatorial warm climates throughout the world (12). Outdoor workers have considerably greater exposure than indoor workers to ambient temperature and to daylight. Yet the extent of the seasonal changes in semen quality among indoor and outdoor workers after adjustment for potential confounding characteristics was remarkably similar. The power of the study was sufficient to detect with statistical significance only a large difference between the groups in summer sperm concentration or in the change in sperm concentration between summer and winter. The results, nevertheless, do not support the hypothesis that it is the heat of the summer that is detrimental to male reproductive capacity. Moreover, we found no correlation between the number of hours worked outdoors during the summer and summertime measures of semen quality or the differences in these measures between summer and winter. In contrast to men who work indoors, outdoor workers are also exposed to a much greater intensity of light during the summer than in other seasons. Absence of a detectable difference in semen quality between indoor and outdoor workers, therefore, suggests also that seasonal variation in light intensity is not the cause of the seasonal variation in semen quality. Evidence from the literature supports the view that summer heat has little or no effect on semen quality (13). The similar study of outdoor workers in San Antonio found no relationship between the number of hours worked outdoors during the summer and the summertime sperm concentration or the summer-winter concentration difference (2). Reports from Edinburgh, United Kingdom (14); Lille, France (15); Basel, Switzerland (16); and Calgary, Canada (1) indicate that the summer sperm concentration may be reduced also in cooler climates. Furthermore, there is little difference between the extent of the summer reduction in sperm concentration noted in studies performed in central or northern Europe or in Canada (cooler climates) Levine et al.
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and in those conducted at lower latitudes (warmer climates) of the northern hemisphere (1). An effect of photoperiod (the length of daylight) on semen quality should be considered. Even relatively brief, periodic exposure to daylight may be sufficient to entrain circannual rhythms of the human biological clock if illumination occurs at the appropriate time in the circadian cycle. For example, administration of light for treating seasonal affective disorder, a recurrent depressive illness commonly occurring in the winter, is said to be more effective in the morning than in the evening (17). Moreover, the same light pulse falling on a mouse either early or late in the subjective night or during the day may respectively delay or advance the phase of the circadian system or have no effect (18). Although indoor workers have less exposure to daylight than outdoor workers, they may nonetheless receive sufficient stimulation to set an internal seasonal clock. Studies in laboratory animals have demonstrated that the suprachiasmatic nuclei ofthe hypothalamus serve as the principal circadian pacemaker in mammals. Specialized photo receptors in the retina are connected directly to these nuclei by the retinohypothalamic tract (18). It is hypothesized that the suprachiasmatic nuclei may modulate the secretion of melatonin by the pineal gland. This in turn could influence the release of pituitary gonadotropins, possibly by controlling the discharge of hypothalamic gonadotropin-releasing hormone (19). A recent study of serum testosterone (T) concentrations in 4,462 United States military veterans found a seasonal pattern with highest levels in the winter and lowest in the summer (20). This pattern appears to be consistent with a neuroendocrine role of photoperiod in causing seasonal variation in sperm production and semen quality. If, alternatively, summer heat directly damaged the seminiferous tubules or epididymis, it is unlikely that this mild heat stress would also affect the Leydig cells, which are relatively resistant to heat-induced damage (21). After injury to the testes, therefore, the level of circulating gonadotropins and perhaps also ofT might be expected to increase, but not decrease, during the summer season in response to feedback mechanisms. In certain mammalian species, changes in the light-dark cycle have been shown to cause profound effects on male reproductive function (22-24). The testis size of rhesus monkeys was increased after exposure to short days (8 hours of light, 16 hours of dark) but decreased after long days (16 hours oflight, 8 hours of dark), with concommitant increases and 1082
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Air conditioning and semen quality
decreases in plasma T, respectively (25). This pattern in a nonhuman primate fits observations of wintertime increases and summertime decreases in human semen quality and T. In summary, the available evidence suggests that photoperiod rather than heat may be the cause of the seasonal changes in semen quality. Except in populations living near the equator, these changes would be expected to occur worldwide. Acknowledgments. We thank David Mortimer, Ph.D., (Sydney IVF Pty Ltd, Sydney, Australia) and Ravi Mathew, M.S., (Department of Biostatistics, University of North Carolina, Chapel Hill, NC) for discussions and assistance. We are grateful to David O. Clarke, Ph.D., and Dragana A. Andjelkovich, M.D., for reviewing the draft manuscript, and to Fred Miller, Ph.D., (all three at Chemical Industry Institute of Toxicology, Research Triangle Park, NC) for making personnel and facilities available for revision of the manuscript.
REFERENCES 1. Levine RJ. Seasonal variation in human semen quality. In: Zorgniotti, AW, editor. Temperature and environmental effects on the testis. New York: Plenum Press, 1991:89-96. 2. Levine RJ, Mathew RM, Chenault CB, Brown MH, Hurtt ME, Bentley KS, et al. Differences in the quality of semen in outdoor workers during summer and winter. N Engl J Med 1990;323:12-6. 3. National Oceanic and Atmospheric Administration; National Environmental Satellite, Data, and Information Service; National Climatic Data Center. Climatological data, Louisiana. (Publication no. C55.214/17). Washington, DC: Government Printing Office, 1989, vol. 94; June, July, and December and 1990, vol. 95; January. 4. Steel RGD, Torrie JH. Principles and procedures of statistics: a biometrical approach. 2d ed. New York: McGraw-Hill, 1980. 5. Cohen J. Statistical power analysis for the behavioral sciences. rev. ed. New York: Academic Press, 1977. 6. Milliken GA, Johnson DE. Analysis of messy data. Vol. 1. designed experiments. New York: Van Nostrand Reinhold, 1984:106-7. 7. Goodnight JH, Harvey WR. Least squares means in the fixed effects general linear model: SAS technical report R-103. Cary (NC): SAS Institute, 1978:8. 8. U.S. Department of Labor, Employment and Training Administration. Dictionary of occupational titles. 4th ed. Washington, DC, 1977. 9. Levine RJ, Bordson BL, Mathew RM, Brown MH, Stanley JM, Starr TB. Deterioration of semen quality during summer in New Orleans. Fertil Steril1988;49:900-7. 10. MacLeod J, Gold RZ. The male factor in fertility and infertility. II. Spermatozoon counts in 1,000 men of known fertility and in 1,000 cases of infertile marriage. J Urol 1951;66: 436-49. 11. Steinberger E, Rodriguez-Rigau LJ. The infertile couple. J AndroI1983;4:1l1-8. 12. Becker S. Seasonal patterns of births and conception throughout the world. In: Zorgniotti AW, editor. Temperature and environmental effects on the testis. New York: Plenum Press, 1991:59-72.
Fertility and Sterility
13. Snyder PJ. Fewer sperm in the summer-it's not the heat, it's . . . N Engl J Med 1990;323:54-6. 14. Mortimer D, Templeton AA, Lenton EA, Coleman RA. Annual patterns of human sperm production and semen quality. Arch AndroI1983;10:1-5. 15. Saint Pol P, Beuscart R, Leroy-Martin B, Hermand E, Jablonski W. Circannual rhythms of sperm parameters of fertile men. Fertil Steril 1989;51:1030-3. 16. Politoff L, Birkhauser M, Almendral A, Zorn A. New data confirming a circannual rhythm in spermatogenesis. Fertil Steril 1989;52:486-9. 17. Terman M, Terman JS, Quitkin FM, McGrath PJ, Stewart JW, Rafferty B. Light therapy for seasonal affective disorder-a review of efficacy. Neuropsychopharmacology 1989;2: 1-22. 18. Moore-Ede MC, Czeisler CA, Richardson GS. Circadian timekeeping in health and disease. N Engl J Med 1983;309: 469-76. 19. Lincoln GA, Short RV. Seasonal breeding: nature's contraceptive. Recent Prog Horm Res 1980;36:1-52.
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20. Dabbs JM Jr. Age and seasonal variation in serum testosterone concentration among men. Chronobiol Int 1990;7: 245-9. 21. Kandeel FR, Swerdloff RS. Role of temperature in regulation of spermatogenesis and the use of heating as a method for contraception. Fertil SteriI1988;49:1-23. 22. Moore-Ede MC, Sulzman FM, Fuller CA. The clocks that time us: physiology of the circadian timing system. Cambridge (MA): Harvard University Press, 1982:288-92. 23. Lincoln GA. Seasonal aspects of testicular function. In: Burger H, de Kretser, D, editors. The Testis. New York: Raven Press, 1981:255-302. 24. Gwinner E. Circannual rhythms-endogenous annual clocks in the organization of seasonal processes. New York: SpringerVerlag Publishers, 1986. 25. Chik CL, Almeida OFX, Libre EA, Booth JD, Renquist D, Merriam GR. Photoperiod-driven changes in reproductive function in male rhesus monkeys. J Clin Endocrinol Metab. In press.
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Vol. 57, No.5, May 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Air-conditioned environments do not prevent deterioration of human semen quality during the summer*
Richard J. Levine, M.D.t:j: Michelle H. Brown, B.S.P.H.t Michelle Bell, M.S.§ II
Frances Shue, M.D.§ Gary N. Greenberg, M.D.~ Brenda L. Bordson, Ph.D.§
Chemical Industry Institute of Toxicology, Research Triangle Park; Duke University Medical Center, Durham, North Carolina; Reproductive Resources Inc., Metairie; and Tulane University, New Orleans, Louisiana
Objective: To determine if air conditioning might mitigate summer reductions in semen quality. Design: Prospective study of semen quality in summer and winter. Setting: Normal human volunteers were studied in the setting of a fertility laboratory. Patients, Participants: Two groups of volunteers were selected from the vicinity of New Orleans: 64 men who worked indoors during the summer in air-conditioned environments and 76 others who worked outdoors. Interventions: None. Main Outcome Measures: Parameters of manual semen analysis were examined for seasonal and group differences. Results: Remarkably similar reductions in semen quality during summer as compared with winter were observed in both indoor and outdoor workers, respectively, with regard to the following parameters of semen quality: 19% and 19% in sperm concentration, 25% and 27% in total sperm per ejaculate, 17% and 20% in motile sperm concentration, 13% and 15% in percent sperm with normal morphology, and 23% and 23% in concentration of morphologically normal motile sperm. Conclusions: These findings do not support the hypothesis that the heat of the summer is detrimental to male reproductive capacity. The available evidence suggests instead a possible role of photoperiod in causing the seasonal changes in semen quality. Fertil Steril 1992;57:1075-83 Key Words: Semen, sperm, summer, winter, heat, photoperiod, air-conditioning
Human sperm concentration, total sperm count per ejaculate, and motile sperm concentration are
Received March 15, 1991; revised and accepted January 8, 1992. * Supported by the Chemical Industry Institute of Toxicology; Duke University Medical Center, Division of Occupational and Environmental Medicine; and Reproductive Resources Inc. t Chemical Industry Institute of Toxicology. t Reprint requests and present address: Richard J. Levine, M.D., Epidemiology Branch; Division of Epidemiology, Statistics, and Prevention Research; National Institute of Child Health and Human Development; National Institutes of Health, EPN 640, Bethesda, Maryland 20892. § Reproductive Resources Inc. II Department of Cellular and Molecular Biology, Tulane University. ~ Division of Occupational and Environmental Medicine, Duke University Medical Center. Vol. 57, No.5, May 1992
reduced during the summer season (1, 2). Could the heat of the summer cause the quality of semen to deteriorate? If so, men who work outdoors in a warm climate during the middle of the day should exhibit a greater decline in sperm concentration than those who work indoors in air-conditioned environments. We report here the results of a prospective study of semen quality during July to August 1989 and January to February 1990 among indoor and outdoor workers in New Orleans (average maximal daily temperature 31.9°C in June and July 1989 and 17.0°C in December 1989 and January 1990) (3). MATERIALS AND METHODS Subjects
Men who worked indoors or outdoors during the summer were recruited in the summer of 1989 Levine et al.
Air conditioning and semen quality
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through newspaper advertisements and asked to visit a reproductive laboratory once that summer and once the following winter. At each visit the men were interviewed, and they provided fresh semen specimens. Summer visits took place within the period July 15 to August 16, 1989 (median, July 31 for indoor workers, July 27 for outdoor workers); winter visits occurred within the period January 12 to February 16, 1990 (median, January 26 for both indoor and outdoor workers). All men gave informed consent for the study and were paid for their participation. Outdoor workers were defined as persons who worked outdoors during summer at least 4 hours per day between 8:00 A.M. and 6:00 P.M. All other dayshift workers were considered to be indoor workers except persons who worked indoors in warm environments (e.g., cook, baker, lifeguard at an indoor swimming pool), who were excluded from the study. Of 224 volunteers who visited the laboratory during the summer, 178 (76 indoor, 102 outdoor workers) were asked to return the next winter. The others were discarded for the following reasons: in collecting semen specimens 19 reported a loss of seminal fluid exceeding a drop; 12 were judged to be unreliable informants or of an undesirable character; 9 could not be classified as either indoor or outdoor workers; and 6 did not produce suitable semen specimens. A total of 142 men (65 indoor, 77 outdoor workers), or 80% of those requested, did in fact return during the winter. Satisfactory winter specimens were obtained from all of these, but 3 men reported a collection loss greater than a drop and were required to provide a substitute specimen a week later. Questionnaires
At each laboratory visit the subjects were asked how long it had been since their most recent ejaculation «24 hours or a specific number of days), whether during the previous 3 months they had had any febrile illness or used any medications (and if so, the medications used), and whether they had collected their specimens completely. Information was obtained about the type of work performed, the duration of employment, the usual time for starting and finishing work, the number of hours per day at work spent outdoors or in air-conditioned settings within the past 3 months, and the number of hours per day spent outdoors when not at work. Subjects were also questioned about the presence of any urethral discharge or dysuria within the past month or any testicular pain within the past 3 months, con1076
Levine et a1.
Air conditioning and semen quality
sumption of alcoholic beverages during the past 3 months, and cigarette smoking history including the number of cigarettes smoked per day during the past 3 months. Laboratory Methods
Before semen collection, subjects were requested to abstain from ejaculation for at least 2 days. Fresh ejaculates were produced at the laboratory by masturbation into sterile plastic containers. Specimens were allowed to liquefy at room temperature. If liquefaction was incomplete, semen analysis was begun 45 minutes after specimen collection. Laboratory analysis of semen volume and sperm count and motility was performed by one laboratory technician. This technician also made an initial assessment of sperm morphology from stained slides shortly after each specimen had been obtained. When all specimens had been collected, identifying information was concealed, and slides were thoroughly shuffled. Then a second, more experienced observer, "blinded" with regard to season and subject, read the slides for sperm morphology. Only the quantitative data on sperm morphology obtained by the blinded, more experienced observer is reported here. Later another experienced observer, also blinded, read the slides again for sperm morphology. After the determination of seminal fluid volume with a graduated syringe, the concentration of spermatozoa was measured with a Neubauer hemocytometer (Reichert-Jung Leica Inc., Buffalo, NY), using a 1:20 dilution of semen to diluent. To arrive at sperm concentration of X106/mL, sperm with tails were counted in 5 of the 25 large squares in the central portion of the hemocytometer. Those on the edge of a square were included in the count if the greater portion of the sperm head fell within the square. Total sperm per ejaculate was the product of sperm concentration and semen volume. For motility assessment, a wet preparation was made by placing a 15-ttL drop of undiluted semen between a slide and a coverslip. A total of 100 sperm (or 4 to 6 microscopic fields) was scanned using a phase-contrast microscope. The motility of each sperm was classified as follows: 0, immotile; 1, nonprogressively motile; 2, weak progression; 3, moderate progression; 4, excellent progression. Percent motile sperm was computed as the percentage of sperm with motility grades 1 to 4. The motility grade of the motile sperm in a specimen was determined by multiplying the number of sperm in each category of motile sperm by the score assigned to that cateFertility and Sterility
gory (1, 2, 3, or 4) and dividing by the total number of motile sperm. To evaluate sperm morphology, a drop of fresh semen was smeared on a slide, air dried, fixed with ethanol, and stained using Gram crystal violet and Gram safranin. One hundred sperm were assigned to the following categories: small head, large head, two heads, two tails, tapered head, coiled tail, bent midpiece, cytoplasmic droplet present, pyriform head, amorphous head, or normal (i.e., absence of the foregoing characteristics). Statistical Analysis
Number of Subjects and Specimens
Of the 142 subjects who provided specimens in both summer and winter, 2 (1 indoor, 1 outdoor worker) who were azoospermic in both seasons were excluded because seasonal variation in semen quality cannot be detected in men without sperm. Three indoor workers who had been laboratory sperm donors before the study and were known to be highly fertile were removed from the comparisons of indoor and outdoor workers. The participation of these subjects had been solicited and was not in response to the newspaper advertisements. Had they been included in the between-group comparisons, aggregate values of the indoor workers would have been elevated unfairly. These three men, however, were retained for the within-person comparisons of semen quality in summer and winter. Because of the likelihood of laboratory error, one indoor worker with an extreme summer-winter concentration difference (summer ~ winter) was omitted from analyses of concentration-related measures. In another indoor worker for whom winter motility data were missing, parameters of sperm motility could not be compared between summer and winter. Slides prepared for sperm morphology had not been retained for the summer specimens of 13 study participants (3 indoor workers, 10 outdoor workers). The winter slide of another subject contained too few sperm to evaluate. After removing these 14 subjects, a total of 126 men with morphological analyses in both summer and winter was used to assess the effect of season on sperm morphology. All 260 specimens remaining after excluding the 3 indoor workers who had been laboratory sperm donors were used in the within-season comparisons of indoor and outdoor workers. The winter specimens of the 13 subjects without summer slides and the summer specimen of the man whose winter slide could not be evaluated were included in these comparisons. Vol. 57, No.5, May 1992
Significance of Paired Seasonal Differences
For comparisons across seasons within subjects, paired t-tests and ANOVA were employed to assess the effect of season on abstinence and on semen quality wherever distributions could be normalized. Arithmetic transformations were needed for all variables except counts of morphologically normal sperm. Cube-root transformations were used for ejaculate volume, sperm concentration, total sperm count per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm. A log transformation was used for abstinence. The distributions of the frequencies of abnormal morphological types could not be normalized by cube root, square root, or log transformations. These variables were, therefore, analyzed with Wilcoxon's signed rank test. This nonparametric method was also applied to the untransformed variables mentioned above. Because it led to the same conclusions as the parametric tests on the normalized data, only results of the latter are reported. Significance of Differences Between Indoor and Outdoor Workers
T-tests were used to determine if arithmetic summer-winter differences in concentration-related parameters of semen quality (sperm concentration, total sperm count, motile sperm concentration, and concentration of morphologically normal motilesperm) varied significantly between indoor and outdoor workers. Analyses of variance were employed to assess the significance of within-season differences in concentration-related variables. Nonparametric analyses of within-season differences between groups were also performed on the untransformed variables, with the Kruskal-Wallis and the van der Waerden (normal scores) tests. Because these yielded the same conclusions as the parametric tests, only the results of the latter are reported. Statistical power calculations were performed using standard methods (4, 5). Control of Confounders
To control for potential confounding characteristics, the ANOV As were enhanced by the addition of measures of possible confounding. The following continuous variables were added as covariates: abstinence (specific number of days, or a half day if <24 hours) in the inverse scale, current smoking (packs of cigarettes per day), and current drinking (drinks per month). Fever and symptoms of possible urogenital disease (urethral discharge, dysuria, and Levine et al.
Air conditioning and semen quality
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Table 1
Characteristics of Seminal Fluid Samples Obtained From 64 Indoor Workers in Summer and Winter Summertime change *
Unadjusted values
Unadjusted %:j:
Adjusted %:j:§
2.5 72.2 171.3 45.1
-2 -18 -25 -14
(-12, 10) (-32, 7) (-38,9) (-23, -5)g
-2 -19 -25 -13
52.6 2.9 41.1 19.8
-2 -1 -15 -22
(-8, 4) (-7,6) (-35,6) (-46,4)'
0(-7,6) 0(-6,7) -17 (-35,13) -23 (-45, 2)"
Variablet
Winter
Summer
Volume of ejaculate (mL) I Sperm concentration (X10 6 jmL) I a Total sperm per ejaculate (X10 6 ) II" Sperm with normal morphology (%)C Motile sperm Percent" Motility grade" Concentration (X10 6 jmL) lib Concentration with normal morphology (X10 6 jmL) lid
2.5 88.3 229.4 52.4 53.7 3.0 48.4 25.5
(-11, (-31, (-36, (-21,
10) 12) 13) -5)g
* Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "63 subjects, b62 subjects, C61 subjects, and d 59 subjects. :j: Two-tailed test for the significance of the seasonal difference; "P < 0.10, 'p < 0.05, and gp < 0.005.
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. I A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
testicular pain) were added as binary variables according to season-specific yes-no categories. Collection losses could not confound the results because none of the specimens used for analysis had collection losses in excess of a drop. Medications were not a problem because no subjects reported use of pharmaceuticals known to affect semen quality, such as anticancer, nitrofuran, or sulfa drugs.
During the summer of 1989, the subjects who were outdoor workers spent an average of 7.5 hid at work outdoors (6.5 hid during winter) and 0.5 hours in air-conditioned surroundings. These circumstances were reversed among indoor workers, who spent 0.5 hid outdoors and 8.5 hid in air-conditioned surroundings. Even when not at work, outdoor workers spent more daytime outdoors than indoor workers (in summer 1 hour versus 0.5 hid during the week, 3.5 hours versus 2 hid on the weekend). Indoor workers were employed largely in professional, technical, and managerial occupations (55%) or in clerical and sales occupations (19%). The most prevalent job categories among outdoor workers were structural work occupations (42%, mostly construction trades, such as welder, electrician, painter, carpenter, brick mason, roofer), agricultural occupations (18%), and miscellaneous occupations (22%) including driving a truck and servicing vehicles (8). The seasonal characteristics of the seminal-fluid samples from the 64 indoor workers, the 76 outdoor workers, and all 140 subjects without azoospermia are presented in Tables 1,2, and 3. The sperm concentration, total sperm count per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm among individual men were lower in summer than in winter. Many of the unadjusted reductions were statistically significant. Reductions for indoor workers (18%, 25%, 15%, and 22%, respectively) were somewhat less than those for outdoor workers (24%,33%,23%, and 25%, respectively), but disparities between the groups with respect to the summer-winter difference for any
Confidence Limits
Two-sided 95% confidence limits (Cl) were determined for the difference between summer and winter for each measure of semen quality, with SEs from the ANOVA (6, 7). Confidence limits were then expressed as a percentage of the winter's mean value. For the analyses conducted in the cube-root scale, the confidence limits are reported after retransformation to the natural scale. RESULTS
Of the 140 subjects without azoospermia who were used in the analysis, 102 were white, 37 were black, and 1 was mixed race of French descent (Creole). The proportion of nonwhite men was similar among indoor and outdoor workers (28% and 26%, respectively). The men ranged from 18 to 57 years of age (mean of 30 for both indoor and outdoor workers). Outdoor workers were more likely than indoor workers to be current smokers (64% versus 36%, respectively) and to consume two or more drinks per day of beer, wine, or spirits (39% versus 13%). 1078
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Air conditioning and semen quality
Fertility and Sterility
Table 2
Characteristics of Seminal Fluid Samples Obtained From 76 Outdoor Workers in Summer and Winter Summertime change'
Unadjusted values Variablet
Winter
Summer
Volume of ejaculate (mL) II Sperm concentration (X10 6 jmL) II Total sperm per ejaculate (X10 6 ) I Sperm with normal morphology (%)" Motile sperm Percent Motility grade Concentration (X10 6 jmL) II Concentration with normal morphology (X10 6 jmL) II"
2.3 73.4 174.5 50.9
2.3 55.8 116.6 43.8
52.8 2.9 41.6 22.3
52.9 2.8 32.1 16.7
Unadjusted %:j:
Adjusted %:j:§
-2 -24 -33 -14
3 -19 -27 -15
(-10, (-34, (-40, (-24,
10) -2)C O)C -4)d
0(-7,7) -3 (-9,4) -23 (-36, l)b -25 (-47, -6)C
(-7, 18) (-30, 10) (-34, 19) (-28, -3)C
0(-8,8) -1 (-8, 7) -20 (-33, 13) -23 (-45,9)
• Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "65 subjects. :j: Two-tailed test for the significance of the seasonal difference; bp < 0.10, cp < 0.05, and dp < 0.005.
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. I A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
measure of semen quality did not achieve significance. There were no significant differences between indoor and outdoor workers in regard to the measures of summer semen quality itself. Morphologically normal sperm decreased in each group by 14% during summer as compared with winter, a change that was highly statistically significant. There were minimal differences in the length of the abstinence period preceding specimen collection between indoor and outdoor workers and between summer and winter seasons. In the combined groups, the values for sperm concentration obtained in winter exceeded the sum-
mer values in 59% of the subjects. However, the proportion of men with sperm concentrations < 20 X 106/mL was lower in summer than in winter (20 of 139 or 14% in summer versus 26 of 139 or 19% in winter), a difference that was not statistically significant. Adjustments were made with linear models for the confounding influence of length of abstinence, cigarette smoking, consumption of alcoholic beverages, symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain), and fever, considered jointly. The magnitude of the summer reduction in sperm concentration, total
Table 3
Characteristics of Seminal Fluid Samples Obtained From 64 Indoor and 76 Outdoor Workers in Summer and Winter Unadjusted values
Summertime change*
Variablet
Winter
Summer
Unadjusted %:j:
Volume of ejaculate (mL) I Sperm concentration (X10 6 jmL) II" Total sperm per ejaculate (X10 6 ) II" Sperm with normal morphology (%)C Motile sperm Percent" Motility grade" Concentration (X10 6 jmL) lib Concentration with normal morphology (X10 6 jmL) lid
2.4 80.1 199.3 51.7
2.4 63.2 141.4 44.4
-2 (-8,7) -21 (-28, -4)f -29 (-34,-W -14 (-20, -8)h
1 -20 -26 -15
53.2 2.9 44.7
52.7 2.9 36.1
-1 (-6,4) -2 (-6,3) -19 (-30, -3)f
-1 (-5,4) 0(-5,4) -19 (-28, 3)·
23.9
18.2
-24 (-41, -12)"
-24 (-40, -7) f
* Changes shown are for summer values as compared with the winter values, as computed from the mean values for summer and winter before rounding. t Determined for "139 subjects, b138 subjects, c126 subjects, and d124 subjects. :j: Two-tailed test for the significance of the seasonal difference; .p < 0.10, fp < 0.02, .p < 0.001, and hp < 0.0001. Vol. 57, No.5, May 1992
Adjusted %:j:§ (-6, 10) (-26, 1)· (-28, 5) (-22, -8)h
§ Adjusted for length of abstinence, cigarette smoking, consumption of alcoholic beverages, fever, and symptoms of possible urogenital disease (urethral discharge, dysuria, and testicular pain). Values in parentheses are 95% CI. II A cube root transformation was used to test the significance of the seasonal effect and to develop 95% CI.
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Air conditioning and semen quality
1079
sperm per ejaculate, motile sperm concentration, and concentration of morphologically normal motile sperm was altered little after such adjustments (Tables 1, 2, and 3), but differences in the extent of reduction between indoor and outdoor workers narrowed (19%, 25%, 17%, and 23% among indoor workers versus 19%, 27%, 20%, and 23% among outdoor workers, respectively). After adjustment, the concentration of morphologically normal motile sperm continued to be significantly reduced during summer among all subjects, and the percentage of morphologically normal cells remained significantly lower in summer in both groups of workers. Besides season and abstinence, no variable explained > 1 % of the conditional within -subject variance of the concentration-related measures and the percentage of morphologically normal cells. The power of this study to detect any but a large difference between indoor and outdoor workers in sperm concentration was small. The true summer sperm concentration among outdoor workers must be at least 41 % less than that of indoor workers for there to have been 80% likelihood of observing a significant difference; a much larger percent increase in the summer-winter difference in sperm concentration is needed among outdoor workers for their seasonal variation to have been as likely to exceed that of indoor workers significantly. Analyses of variance with or without confounding variables revealed no significant correlations between the number of hours per day spent outdoors at work during the summer and the summer values of concentration-related measures of semen quality and the percentage of sperm with normal morphology. There were no significant correlations with the differences between summer and winter values. During the initial unblinded assessment of sperm morphology, the percentage of sperm with normal morphological features was found to be significantly greater during the summer. A winter increase in the frequency of sperm with cytoplasmic droplets accounted for most of the difference. A significant summer increase in the frequency of sperm with tapered heads was also observed. After the completion of specimen collection when all slides were read by the first experienced, blinded observer, a highly significant decrease in the proportion of sperm with normal morphology was found in the summer. This resulted from significant (or near significant) summer increases in the proportion of sperm with ta~ pered or pyriform heads, detected in both indoor and outdoor workers. Sperm with cytoplasmic droplets were found to be slightly more frequent during 1080
Levine et al.
Air conditioning and semen quality
the summer. The second experienced, blinded observer also observed a highly significant (P < 0.004) decrease in the proportion of sperm with normal morphology during summer. This observer found significant summer increases in sperm with small, tapered, or amorphous heads and in sperm with cytoplasmic droplets. DISCUSSION
We conducted pairwise analyses of semen specimens obtained in summer and winter from men who worked indoors in air-conditioned environments or outdoors during the summer in the vicinity of New Orleans. Substantial summer reductions of sperm concentration, total sperm per ejaculate, motile sperm concentration, percent sperm with normal morphology, and concentration of morphologically normal motile sperm were revealed among both groups of workers. After adjustment for potential confounding factors, seasonal differences remained substantial but were statistically significant within each group only for percent sperm with normal morphology. Because adjusted summer-winter differences were similar in indoor and outdoor workers, the groups could logically be combined to provide a more powerful test of the overall seasonal effect in New Orleans workers. After adjustment, summer reductions in most concentration-related measures of semen quality and in the percentage of sperm with normal morphology were significant or of borderline significance in the combined groups. The results confirm the deterioration in sperm concentration and related parameters of semen quality that has been noted repeatedly during the summer at various nonequatorial locations in the northern hemisphere (1). Moreover, they support the finding of a summer reduction in the percentage of sperm with normal morphological features, previously reported in a retrospective study of patients at a New Orleans fertility clinic (9). In that study, among 1,159 samples contributed by 903 men, there were significantly fewer sperm with normal morphological features during summer than in fall, winter' and spring. However, this was not observed in pairwise comparisons among the 61 men who had provided specimens in both summer and other seasons. A significant summer excess of sperm with bent midpieces was detected among all samples, whereas in the present study, sperm with tapered or pyriform heads were found to be significantly increased. Differences between morphology assessments made by the first technician and the others were Fertility and Sterility
striking. The first technician was less experienced, was aware of the season in which the specimens had been obtained, and analyzed slides in the order in which they had been made, with a gap of 6 months separating analyses of the summer and winter samples. It may have been difficult, therefore, for this technician to maintain consistency across seasons in the subjective procedure of reading slides. This could have resulted in a more critical approach taken toward the winter specimens. Besides being more experienced, the second and third technicians were blinded as to season as well as subject and completed all analyses in random order within 2 to 3 weeks. All three technicians reported a significant excess of tapered sperm heads during the summer season. Observer bias across seasons may have influenced the measurements of sperm motility and progression. Because these analyses require fresh semen, it was not possible to blind the laboratory technician with respect to season. Despite the potential for bias, no seasonal difference was observed in the percentage of motile sperm or in motility grade. A similar study in San Antonio in which automated semen analyses had been performed with a computerized image analysis system, thus reducing the likelihood for bias, found no summer-winter differences in the percentage of motile sperm or in the characteristics of sperm movement (velocity, linearity, linear velocity, mean amplitude of lateral head displacement, and beat/cross frequency) (2). Values for semen volume and sperm concentration, which were determined from fresh semen through more objective means, are less likely to be biased. In assessing sperm concentration, the component of the measurement process most susceptible to observer inconsistency would appear to be judging whether sperm with heads on the edge of a square should be included in the count. However, even if all sperm on the edges of the squares were included for the winter specimens (equivalent to enlarging each of the squares' two dimensions by the length of a sperm head) and none for the summer, the resulting difference can be calculated to be <5%, far less than the 21 % actually observed. A summertime increase in the proportion of men with sperm concentrations < 20 X 106/mL (oligospermia) and considered at higher risk of infertility (10) was not detected in this study. The similar study of 131 men in San Antonio had found oligospermia to be considerably more frequent during the summer (2). Although the two studies involved comparable numbers of subjects, in the latter study sperm concentrations in each season were usually determined Vol. 57, No.5, May 1992
from the mean of two specimens. Seasonal values for sperm concentration, therefore, were more stable, enhancing the potential for detecting nonrandom change. Besides increasing the risk of infertility and the chance that pregnancy will not occur, deterioration of semen quality in a population may affect fertility rates by lengthening the time to pregnancy (11). It is possible that the summer reduction in semen quality may contribute to the deficit in spring births that occurs in nonequatorial warm climates throughout the world (12). Outdoor workers have considerably greater exposure than indoor workers to ambient temperature and to daylight. Yet the extent of the seasonal changes in semen quality among indoor and outdoor workers after adjustment for potential confounding characteristics was remarkably similar. The power of the study was sufficient to detect with statistical significance only a large difference between the groups in summer sperm concentration or in the change in sperm concentration between summer and winter. The results, nevertheless, do not support the hypothesis that it is the heat of the summer that is detrimental to male reproductive capacity. Moreover, we found no correlation between the number of hours worked outdoors during the summer and summertime measures of semen quality or the differences in these measures between summer and winter. In contrast to men who work indoors, outdoor workers are also exposed to a much greater intensity of light during the summer than in other seasons. Absence of a detectable difference in semen quality between indoor and outdoor workers, therefore, suggests also that seasonal variation in light intensity is not the cause of the seasonal variation in semen quality. Evidence from the literature supports the view that summer heat has little or no effect on semen quality (13). The similar study of outdoor workers in San Antonio found no relationship between the number of hours worked outdoors during the summer and the summertime sperm concentration or the summer-winter concentration difference (2). Reports from Edinburgh, United Kingdom (14); Lille, France (15); Basel, Switzerland (16); and Calgary, Canada (1) indicate that the summer sperm concentration may be reduced also in cooler climates. Furthermore, there is little difference between the extent of the summer reduction in sperm concentration noted in studies performed in central or northern Europe or in Canada (cooler climates) Levine et al.
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and in those conducted at lower latitudes (warmer climates) of the northern hemisphere (1). An effect of photoperiod (the length of daylight) on semen quality should be considered. Even relatively brief, periodic exposure to daylight may be sufficient to entrain circannual rhythms of the human biological clock if illumination occurs at the appropriate time in the circadian cycle. For example, administration of light for treating seasonal affective disorder, a recurrent depressive illness commonly occurring in the winter, is said to be more effective in the morning than in the evening (17). Moreover, the same light pulse falling on a mouse either early or late in the subjective night or during the day may respectively delay or advance the phase of the circadian system or have no effect (18). Although indoor workers have less exposure to daylight than outdoor workers, they may nonetheless receive sufficient stimulation to set an internal seasonal clock. Studies in laboratory animals have demonstrated that the suprachiasmatic nuclei ofthe hypothalamus serve as the principal circadian pacemaker in mammals. Specialized photo receptors in the retina are connected directly to these nuclei by the retinohypothalamic tract (18). It is hypothesized that the suprachiasmatic nuclei may modulate the secretion of melatonin by the pineal gland. This in turn could influence the release of pituitary gonadotropins, possibly by controlling the discharge of hypothalamic gonadotropin-releasing hormone (19). A recent study of serum testosterone (T) concentrations in 4,462 United States military veterans found a seasonal pattern with highest levels in the winter and lowest in the summer (20). This pattern appears to be consistent with a neuroendocrine role of photoperiod in causing seasonal variation in sperm production and semen quality. If, alternatively, summer heat directly damaged the seminiferous tubules or epididymis, it is unlikely that this mild heat stress would also affect the Leydig cells, which are relatively resistant to heat-induced damage (21). After injury to the testes, therefore, the level of circulating gonadotropins and perhaps also ofT might be expected to increase, but not decrease, during the summer season in response to feedback mechanisms. In certain mammalian species, changes in the light-dark cycle have been shown to cause profound effects on male reproductive function (22-24). The testis size of rhesus monkeys was increased after exposure to short days (8 hours of light, 16 hours of dark) but decreased after long days (16 hours oflight, 8 hours of dark), with concommitant increases and 1082
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decreases in plasma T, respectively (25). This pattern in a nonhuman primate fits observations of wintertime increases and summertime decreases in human semen quality and T. In summary, the available evidence suggests that photoperiod rather than heat may be the cause of the seasonal changes in semen quality. Except in populations living near the equator, these changes would be expected to occur worldwide. Acknowledgments. We thank David Mortimer, Ph.D., (Sydney IVF Pty Ltd, Sydney, Australia) and Ravi Mathew, M.S., (Department of Biostatistics, University of North Carolina, Chapel Hill, NC) for discussions and assistance. We are grateful to David O. Clarke, Ph.D., and Dragana A. Andjelkovich, M.D., for reviewing the draft manuscript, and to Fred Miller, Ph.D., (all three at Chemical Industry Institute of Toxicology, Research Triangle Park, NC) for making personnel and facilities available for revision of the manuscript.
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13. Snyder PJ. Fewer sperm in the summer-it's not the heat, it's . . . N Engl J Med 1990;323:54-6. 14. Mortimer D, Templeton AA, Lenton EA, Coleman RA. Annual patterns of human sperm production and semen quality. Arch AndroI1983;10:1-5. 15. Saint Pol P, Beuscart R, Leroy-Martin B, Hermand E, Jablonski W. Circannual rhythms of sperm parameters of fertile men. Fertil Steril 1989;51:1030-3. 16. Politoff L, Birkhauser M, Almendral A, Zorn A. New data confirming a circannual rhythm in spermatogenesis. Fertil Steril 1989;52:486-9. 17. Terman M, Terman JS, Quitkin FM, McGrath PJ, Stewart JW, Rafferty B. Light therapy for seasonal affective disorder-a review of efficacy. Neuropsychopharmacology 1989;2: 1-22. 18. Moore-Ede MC, Czeisler CA, Richardson GS. Circadian timekeeping in health and disease. N Engl J Med 1983;309: 469-76. 19. Lincoln GA, Short RV. Seasonal breeding: nature's contraceptive. Recent Prog Horm Res 1980;36:1-52.
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20. Dabbs JM Jr. Age and seasonal variation in serum testosterone concentration among men. Chronobiol Int 1990;7: 245-9. 21. Kandeel FR, Swerdloff RS. Role of temperature in regulation of spermatogenesis and the use of heating as a method for contraception. Fertil SteriI1988;49:1-23. 22. Moore-Ede MC, Sulzman FM, Fuller CA. The clocks that time us: physiology of the circadian timing system. Cambridge (MA): Harvard University Press, 1982:288-92. 23. Lincoln GA. Seasonal aspects of testicular function. In: Burger H, de Kretser, D, editors. The Testis. New York: Raven Press, 1981:255-302. 24. Gwinner E. Circannual rhythms-endogenous annual clocks in the organization of seasonal processes. New York: SpringerVerlag Publishers, 1986. 25. Chik CL, Almeida OFX, Libre EA, Booth JD, Renquist D, Merriam GR. Photoperiod-driven changes in reproductive function in male rhesus monkeys. J Clin Endocrinol Metab. In press.
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