Association of Sleep Disturbances With Reduced Semen Quality
Association of Sleep Disturbances With Reduced Semen Quality
Because of the military draft in Denmark, all 18-year-old men, except those suffering from severe chronic disease, are required to undergo a compulsory physical examination to determine their fitness for military service. Some men postpone their examination until completion of their education. Since 1996, trained staff from the Department of Growth and Reproduction at Copenhagen University Hospital (Rigshospitalet, Copenhagen, Denmark) have approached the draftees when they have appeared for their compulsory physical examination and have invited them to participate in a study of semen quality. Men recruited from January 2008 to June 2011 were included in the present study, since the questionnaire they completed included detailed information about sleep disturbances. All participants completed a questionnaire, delivered a semen sample, had a blood sample drawn, and underwent a physical examination. They received compensation for their time (kr500, equal to approximately US$85). Participants did not differ from nonparticipants with regard to age, but they were generally better educated than nonparticipants (data not shown). Ethical approval was obtained from the local ethical committee. A detailed description of the study has previously been published.
All men provided a semen sample by means of masturbation in a room close to the semen laboratory. The period of ejaculation abstinence (time since last ejaculation) was recorded, and the semen sample was analyzed for volume, sperm concentration, total sperm count, percent motile spermatozoa, and percent morphologically normal spermatozoa as described by Jørgensen et al., which is in accordance with the most recent guideline from the World Health Organization. Since 1996, our laboratory has led a quality control program for assessment of sperm concentration; the laboratory has kept the interlaboratory difference unchanged, and the variation between technicians was less than 10%. The same 2 experienced technicians assessed sperm morphology according to strict criteria for the first 838 men.
Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone, and sex hormone-binding globulin (SHBG) were determined using a time-resolved immunofluorometric assay (Delfia; Wallac Oy, Turku, Finland). Testosterone and estradiol levels were determined using time-resolved fluoroimmunoassays (Delfia; Wallac Oy). Inhibin B level was determined by means of a specific 2-sided enzyme immunometric assay (Inhibin B Gen II; Beckman Coulter Ltd., High Wycombe, United Kingdom). Intra- and interassay coefficients of variation for measurements of FSH and luteinizing hormone were 3% and 4.5%, respectively. Coefficients of variation for testosterone and SHBG were <8% and <5% respectively. The intraassay coefficients of variation for estradiol and inhibin B were <4% and <7%, respectively, and the interassay coefficients of variation were <4% and <6%, respectively. The hormones were all measured within same time period and in the same assay batches. Therefore, even though some variation in analyses over time may have been present, this would not have affected the results. At the time of submission of this article, reproductive hormone levels had been measured in the first 672 men. Free testosterone was calculated on the basis of the measured serum concentrations of total testosterone and SHBG using the method of Vermeulen et al. and a fixed albumin concentration of 0.62 mmol/L. Because changes in the concentration of albumin have only minor effects on the ratio of total testosterone to free testosterone, it is justifiable to use a fixed mean albumin concentration when individual albumin measurements are not available, provided that there is no reason to suspect significantly abnormal albumin levels. In addition, the ratios between inhibin B and FSH, testosterone and estradiol, and calculated free testosterone and luteinizing hormone were calculated.
Physicians assessed Tanner stage of pubic hair and genital development, testicular volumes (determined using a Prader wooden orchidometer (Pharmacia & Upjohn, London, United Kingdom)), the possible presence of a varicocele (grades 1–3) or hydrocele, and the location of the testes in the scrotum, and the consistency of the testis and epididymis were recorded. Weight and height was measured, and body mass index (BMI) was calculated as weight in kilograms divided by squared height in meters. Conditions detected at the physical examination that may affect semen quality (varicocele grade 2 or 3 or abnormal position of the testes) were summarized in a single variable designated "conditions found at the physical examination."
Prior to the examination, all participants completed a questionnaire that collected information on previous and/or current diseases and genital diseases such as inguinal hernia, varicocele, epididymitis, gonorrhea, chlamydia, and surgery for testicular torsion. The participants were asked whether they had been born with both testicles in the scrotum. In addition, they reported whether they had had fever greater than 38°C (100.4°F) within the previous 3 months and whether they had ever been diagnosed by a physician with depression. Self-reported diseases of the reproductive organs affecting semen quality (torsion of testes, epididymitis, or inguinal hernia) were transformed into 2 variables: "self-reported genital conditions" and "sexually transmitted diseases" (gonorrhea or chlamydia).
Sleep disturbances were assessed on the basis of a modified 4-item version of the Karolinska Sleep Questionnaire (15, 16), which includes questions on sleep patterns during the past 4 weeks: How often have you 1) "slept badly or restlessly"; 2) "found it difficult to fall asleep"; 3) "woken up too early in the morning and not been able to go back to sleep"; and 4) "woken up several times during the night and found it difficult to go back to sleep"? The response categories were "all the time" (scoring 100%), "a large part of the time" (scoring 67%), "rarely" (scoring 33%), and "never" (scoring 0%) and were scored according to the Copenhagen Psychosocial Questionnaire. Because responses to the 4 questions were correlated (ρ = 0.25–0.57), a sleep score was created as the mean of the percentages answered in the 4 questions. Thirteen men did not respond to all of the sleep questions, and a score could not be computed for these men.
The young men responded to a standard questionnaire about maternal education, which was coded as below 9 years of schooling, 9–10 years of schooling, and more than 10 years of schooling. Participants were asked: "How much of the following beverages did you consume during the last week?" Possible responses were: bottles of cola (0.5 L), other sodas (0.5 L), diet cola (0.5 L), other diet sodas (0.5 L), caffeine-containing energy drinks (0.25 L), and number of chocolate bars (50 g). In addition, participants were asked how many cups of coffee, tea, and chocolate-containing beverages they had consumed daily during the last week. Each man's daily caffeine intake was estimated assuming that a cup contained 150 mL and that caffeine content was 117 mg in 1 cup of coffee, 70 mg in 1 cup of tea, 5 mg in 1 cup of chocolate beverages, 70 mg in a 0.5-L cola or diet soft drink, 117 mg in 1 energy drink, and 7 mg in a 50-g chocolate bar. The men were informed that 1 beer, 1 glass of wine, or 40 mL of spirits contained 1 unit of alcohol, whereas 1 strong beer or 1 alcopop contained 1.5 units of alcohol and 1 bottle of wine contained 6 units of alcohol. They were asked about their daily unit intakes of red and white wine, beer, strong alcoholic drinks, and alcopops during the last week. Alcohol intake was calculated as the sum of daily reported unit intakes within the last week. Information on physical activity was coded into watts per week according to the method of Craig et al.
Exposure variables were the 4 individual sleep disturbances and a sleep score. Sleep score was calculated as the mean of the participant's replies to the 4 sleep questions: "all the time" (scoring 100%), "a large part of the time" (scoring 67%), "rarely" (scoring 33%), or "never" (scoring 0%) and was categorized as 0, 1–10, 11–20 (reference), 21–30, 31–40, 41–50, or >50. The category 11–20 was used as the reference category because this group of men had the highest sperm count. First, semen quality, testis size, and reproductive hormone levels were compared for men in relation to sleep disturbances and sleep score categorized as 0, 1–20, 20–40, or >40. Then we compared the distributions of the variables from the questionnaires and physical examinations among men with different sleep disturbance scores by χ test in order to identify potential confounders. Finally, data were analyzed using multiple linear regression analyses. Normally distributed outcome variables were entered directly into the model as continuous variables.
Because of the nonnormal (skewed) distributions of sperm concentration, total sperm count, calculated free testosterone, and FSH and increasing variances, they were analyzed on a natural logarithmic scale and back-transformed to obtain the percentage of change in these parameters. Covariates initially included factors possibly associated with semen parameters, reproductive hormone levels, or sleep score and were then excluded stepwise if they did not change the estimate by more than 10%. The same set of confounders was used for all semen parameters: period of abstinence (transformed by natural logarithm), alcohol intake, smoking, and age and, for sperm motility, also duration between the time of ejaculation and analysis of the sample, categorized as shown in Table 1. The analyses of reproductive hormones were adjusted for time of blood sampling, smoking, and BMI. Tests for trend were performed by inserting the categorical sleep variable into the model, assuming the association to be linear.
Because depression may cause sleep disturbances, we repeated the analyses after excluding the 21 men who had ever had depression diagnosed by a physician. Because the association between sleep disturbances and semen quality and reproductive hormone levels may be mediated through alcohol consumption and smoking habits, we repeated the analyses without adjustment for these factors. In addition, because being overweight may lead to sleep disturbances, we evaluated whether BMI modified the effect by stratifying results according to BMI. The results are presented as regression coefficients with 95% confidence intervals. We evaluated the fit of the regression models by testing the residuals for normality and by inspecting the residual plots.
Materials and Methods
Population
Because of the military draft in Denmark, all 18-year-old men, except those suffering from severe chronic disease, are required to undergo a compulsory physical examination to determine their fitness for military service. Some men postpone their examination until completion of their education. Since 1996, trained staff from the Department of Growth and Reproduction at Copenhagen University Hospital (Rigshospitalet, Copenhagen, Denmark) have approached the draftees when they have appeared for their compulsory physical examination and have invited them to participate in a study of semen quality. Men recruited from January 2008 to June 2011 were included in the present study, since the questionnaire they completed included detailed information about sleep disturbances. All participants completed a questionnaire, delivered a semen sample, had a blood sample drawn, and underwent a physical examination. They received compensation for their time (kr500, equal to approximately US$85). Participants did not differ from nonparticipants with regard to age, but they were generally better educated than nonparticipants (data not shown). Ethical approval was obtained from the local ethical committee. A detailed description of the study has previously been published.
Semen Analysis
All men provided a semen sample by means of masturbation in a room close to the semen laboratory. The period of ejaculation abstinence (time since last ejaculation) was recorded, and the semen sample was analyzed for volume, sperm concentration, total sperm count, percent motile spermatozoa, and percent morphologically normal spermatozoa as described by Jørgensen et al., which is in accordance with the most recent guideline from the World Health Organization. Since 1996, our laboratory has led a quality control program for assessment of sperm concentration; the laboratory has kept the interlaboratory difference unchanged, and the variation between technicians was less than 10%. The same 2 experienced technicians assessed sperm morphology according to strict criteria for the first 838 men.
Serum Samples
Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone, and sex hormone-binding globulin (SHBG) were determined using a time-resolved immunofluorometric assay (Delfia; Wallac Oy, Turku, Finland). Testosterone and estradiol levels were determined using time-resolved fluoroimmunoassays (Delfia; Wallac Oy). Inhibin B level was determined by means of a specific 2-sided enzyme immunometric assay (Inhibin B Gen II; Beckman Coulter Ltd., High Wycombe, United Kingdom). Intra- and interassay coefficients of variation for measurements of FSH and luteinizing hormone were 3% and 4.5%, respectively. Coefficients of variation for testosterone and SHBG were <8% and <5% respectively. The intraassay coefficients of variation for estradiol and inhibin B were <4% and <7%, respectively, and the interassay coefficients of variation were <4% and <6%, respectively. The hormones were all measured within same time period and in the same assay batches. Therefore, even though some variation in analyses over time may have been present, this would not have affected the results. At the time of submission of this article, reproductive hormone levels had been measured in the first 672 men. Free testosterone was calculated on the basis of the measured serum concentrations of total testosterone and SHBG using the method of Vermeulen et al. and a fixed albumin concentration of 0.62 mmol/L. Because changes in the concentration of albumin have only minor effects on the ratio of total testosterone to free testosterone, it is justifiable to use a fixed mean albumin concentration when individual albumin measurements are not available, provided that there is no reason to suspect significantly abnormal albumin levels. In addition, the ratios between inhibin B and FSH, testosterone and estradiol, and calculated free testosterone and luteinizing hormone were calculated.
Physical Examination
Physicians assessed Tanner stage of pubic hair and genital development, testicular volumes (determined using a Prader wooden orchidometer (Pharmacia & Upjohn, London, United Kingdom)), the possible presence of a varicocele (grades 1–3) or hydrocele, and the location of the testes in the scrotum, and the consistency of the testis and epididymis were recorded. Weight and height was measured, and body mass index (BMI) was calculated as weight in kilograms divided by squared height in meters. Conditions detected at the physical examination that may affect semen quality (varicocele grade 2 or 3 or abnormal position of the testes) were summarized in a single variable designated "conditions found at the physical examination."
Questionnaire
Prior to the examination, all participants completed a questionnaire that collected information on previous and/or current diseases and genital diseases such as inguinal hernia, varicocele, epididymitis, gonorrhea, chlamydia, and surgery for testicular torsion. The participants were asked whether they had been born with both testicles in the scrotum. In addition, they reported whether they had had fever greater than 38°C (100.4°F) within the previous 3 months and whether they had ever been diagnosed by a physician with depression. Self-reported diseases of the reproductive organs affecting semen quality (torsion of testes, epididymitis, or inguinal hernia) were transformed into 2 variables: "self-reported genital conditions" and "sexually transmitted diseases" (gonorrhea or chlamydia).
Sleep disturbances were assessed on the basis of a modified 4-item version of the Karolinska Sleep Questionnaire (15, 16), which includes questions on sleep patterns during the past 4 weeks: How often have you 1) "slept badly or restlessly"; 2) "found it difficult to fall asleep"; 3) "woken up too early in the morning and not been able to go back to sleep"; and 4) "woken up several times during the night and found it difficult to go back to sleep"? The response categories were "all the time" (scoring 100%), "a large part of the time" (scoring 67%), "rarely" (scoring 33%), and "never" (scoring 0%) and were scored according to the Copenhagen Psychosocial Questionnaire. Because responses to the 4 questions were correlated (ρ = 0.25–0.57), a sleep score was created as the mean of the percentages answered in the 4 questions. Thirteen men did not respond to all of the sleep questions, and a score could not be computed for these men.
The young men responded to a standard questionnaire about maternal education, which was coded as below 9 years of schooling, 9–10 years of schooling, and more than 10 years of schooling. Participants were asked: "How much of the following beverages did you consume during the last week?" Possible responses were: bottles of cola (0.5 L), other sodas (0.5 L), diet cola (0.5 L), other diet sodas (0.5 L), caffeine-containing energy drinks (0.25 L), and number of chocolate bars (50 g). In addition, participants were asked how many cups of coffee, tea, and chocolate-containing beverages they had consumed daily during the last week. Each man's daily caffeine intake was estimated assuming that a cup contained 150 mL and that caffeine content was 117 mg in 1 cup of coffee, 70 mg in 1 cup of tea, 5 mg in 1 cup of chocolate beverages, 70 mg in a 0.5-L cola or diet soft drink, 117 mg in 1 energy drink, and 7 mg in a 50-g chocolate bar. The men were informed that 1 beer, 1 glass of wine, or 40 mL of spirits contained 1 unit of alcohol, whereas 1 strong beer or 1 alcopop contained 1.5 units of alcohol and 1 bottle of wine contained 6 units of alcohol. They were asked about their daily unit intakes of red and white wine, beer, strong alcoholic drinks, and alcopops during the last week. Alcohol intake was calculated as the sum of daily reported unit intakes within the last week. Information on physical activity was coded into watts per week according to the method of Craig et al.
Statistics
Exposure variables were the 4 individual sleep disturbances and a sleep score. Sleep score was calculated as the mean of the participant's replies to the 4 sleep questions: "all the time" (scoring 100%), "a large part of the time" (scoring 67%), "rarely" (scoring 33%), or "never" (scoring 0%) and was categorized as 0, 1–10, 11–20 (reference), 21–30, 31–40, 41–50, or >50. The category 11–20 was used as the reference category because this group of men had the highest sperm count. First, semen quality, testis size, and reproductive hormone levels were compared for men in relation to sleep disturbances and sleep score categorized as 0, 1–20, 20–40, or >40. Then we compared the distributions of the variables from the questionnaires and physical examinations among men with different sleep disturbance scores by χ test in order to identify potential confounders. Finally, data were analyzed using multiple linear regression analyses. Normally distributed outcome variables were entered directly into the model as continuous variables.
Because of the nonnormal (skewed) distributions of sperm concentration, total sperm count, calculated free testosterone, and FSH and increasing variances, they were analyzed on a natural logarithmic scale and back-transformed to obtain the percentage of change in these parameters. Covariates initially included factors possibly associated with semen parameters, reproductive hormone levels, or sleep score and were then excluded stepwise if they did not change the estimate by more than 10%. The same set of confounders was used for all semen parameters: period of abstinence (transformed by natural logarithm), alcohol intake, smoking, and age and, for sperm motility, also duration between the time of ejaculation and analysis of the sample, categorized as shown in Table 1. The analyses of reproductive hormones were adjusted for time of blood sampling, smoking, and BMI. Tests for trend were performed by inserting the categorical sleep variable into the model, assuming the association to be linear.
Because depression may cause sleep disturbances, we repeated the analyses after excluding the 21 men who had ever had depression diagnosed by a physician. Because the association between sleep disturbances and semen quality and reproductive hormone levels may be mediated through alcohol consumption and smoking habits, we repeated the analyses without adjustment for these factors. In addition, because being overweight may lead to sleep disturbances, we evaluated whether BMI modified the effect by stratifying results according to BMI. The results are presented as regression coefficients with 95% confidence intervals. We evaluated the fit of the regression models by testing the residuals for normality and by inspecting the residual plots.
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