Arch Gynecol Obstet DOI 10.1007/s00404-014-3281-x
Gynecologic Endocrinology and Reproductive Medicine
The impact of cigarette smoking and alcohol consumption on sperm parameters and sperm DNA fragmentation (SDF) measured by Halosperm® G. Anifandis · T. Bounartzi · C. I. Messini · K. Dafopoulos · S. Sotiriou · I. E. Messinis
Received: 20 January 2014 / Accepted: 2 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Both cigarette smoking and alcohol consumption are somehow implicated in sperm function, but the impact of these two lifestyle factors on sperm parameters remains controversial. The present study is focused on the impact of cigarette smoking and alcohol consumption separately and combined on sperm parameters and sperm DNA fragmentation (SDF). Methods The study included 207 consecutive semen samples derived from men who were seeking semen analysis for fertility purposes in our IVF Unit. Results Semen volume, percent of degenerated spermatozoa and SDF were significantly correlated with the various smoking status. The percent of spermatozoa with small halos significantly correlated with the alcohol status. The smoking status of the men was correlated with the alcohol status. Conclusions Cigarette smoking and alcohol consumption separately and combined were found to have deleterious effect on sperm parameters and SDF. It is suggested that both habits may contribute to infertility problems. Keywords Alcohol consumption · Cigarette smoking · DNA fragmentation · Semen quality Introduction Male infertility accounts for approximately 40 % of infertile couples. However, a specific male disorder may not G. Anifandis (*) · T. Bounartzi · C. I. Messini · K. Dafopoulos · S. Sotiriou · I. E. Messinis Department of Obstetrics and Gynaecology, Faculty of Medicine, School of Health Sciences, University of Thessalia, Larisa, Greece e-mail: [email protected]
always be identified and for this reason environmental factors, such as estrogenic pollutants [1], obesity, and lifestyle factors such as cigarette smoking and alcohol intake [2] have drawn much attention. Cigarette smoking and alcohol consumption have been proposed to impact on male fertility and subsequently on reproductive outcome, since the effects that both exert on global and individual health are well known [3]. Nicotine, the main component of cigarettes, has been found to have deleterious effects on sperm parameters [4]. Chronic exposure of spermatozoa on toxic components, such as nicotine or its metabolite cotinine, have been associated with sperm cell death, a situation that is derived after the damage of sperm DNA [5]. When assisted reproduction is used, the outcome is relatively low, because smoking through a molecular mechanism produce spermatozoa with chromosomal abnormalities [6]. Multiple studies have proved that smoking induces alterations in semen including increase in reactive oxygen species (ROS) and decline in antioxidant molecules [7, 8]. It has been reported recently that smoking has a negative effect on sperm parameters, including semen volume, sperm concentration, motility and DNA fragmentation [9–11]. However, other studies failed to find any impact of cigarette smoking on sperm parameters [12, 13]. Several studies focused on the association between alcohol consumption and male fertility. Alcohol intake has been related to reduced sperm parameters including semen volume, sperm count, motility and morphology [14]. Excessive alcohol consumption has been implicated in the alteration of serum hormonal levels, which in turn result in the reduction of seminal plasma fluid [15], underlying a possible inhibitory role of alcohol on gonadotrophin secretion [16]. On the contrary, there are few data that failed to observe any impact of alcohol intake on sperm parameters
13
[17], possibly because most studies are based on the average weekly or monthly alcohol consumption and not on the acute (last 5 days) alcohol intake. Even then Hansen et al. [18] failed to find any significant impact of alcohol on sperm parameters. The impact of smoking and alcohol separately on sperm parameters has been investigated, but with controversial findings. Nevertheless, studies about the effect of both smoking and alcohol consumption are scarce. Moreover, these studies have not reached yet a solid conclusion about the combined impact of both cigarette smoking and alcohol intake and for that reason we undertook this study in order to shed more light relatively on the effect of both factors on sperm parameters and sperm DNA fragmentation (SDF).
Patients and methods Two hundred and seven semen samples obtained by 207 men, for fertility purposes in our Assisted Conception Unit, were prospectively investigated after informed consent and approval of Institutional Review Board. Before semen analysis, a questionnaire was distributed to all men. In the specific brochure, questions concerning their age, height, weight, medical history, smoking habits and alcohol consumption were included. All samples were obtained by masturbation after sexual abstinence of 3–4 days and semen analysis was performed according to 2010 WHO guidelines [19]. In the present study, we evaluated seminal volume, semen concentration, progressive motility (PRM), non-progressive motility (NPR), immotility (IM) and sperm DNA fragmentation index with the Sperm Chromatin Dispersion assay (SCD), known as Halosperm®. Smoking assessment “How many cigarettes have you been smoking (per day) the last year until the current semen sample?” was one of the questions concerning smoking status. The measurement of smoking status was performed according to the type of the pack-year history ranged from 0 to 2.5 pack-year history, which means that a man with a 2.5 pack-year history smokes 2.5 packs of cigarettes per day/per year. The explanatory variable (cigarette smoking) was categorized in three groups: no smokers (n = 98), moderate smokers (0–1 pack-year history, n = 76), heavy smokers (>1 packs-year history, n = 33).
Arch Gynecol Obstet
measurement of alcohol intake was estimated by alcohol units: 1 alcohol unit was considered for 100 ml wine, 200 ml beer, for 30 ml whisky and vodka. The explanatory variable (alcohol intake) was divided into three groups: no alcohol users (n = 112), moderate alcohol users or alcohol users drinking >0 to 7 units/week (n = 25). Assessment of DNA fragmentation Sperm DNA damage was performed with the sperm chromatin dispersion (SCD) assay, known as the Halosperm® G2 kit. Briefly, a maximum of 20 μl of the semen sample was diluted to 20 million/ml in phosphate-buffered saline (PBS). Eppendorf tubes were placed in a water bath at 90– 100 °C for 5 min in order the agarose to be fully melted. After 5 min of incubation, we transfer 50 μl of the diluted semen sample to the 100 μl melted agarose tube and we mix gently with a pipette. We placed immediately 8 μl of the cell suspension onto the sample well and we cover with a coverslip. Slides were then placed on a gold plate in the refrigerator (4 °C) for 5 min in order for the agarose to be solidified with the sperm cells embedded within. After taking out the slides of the fridge, the coverslips were gently removed and the slides were placed horizontally in an elevated position. We then apply with the denaturant agent, making sure the slide is fully covered and we incubate for 7 min. The slides afterwards were horizontally immersed in a lysis buffer and incubated for 20 min. After washing with abundant distilled water for 5 min, the slides were dehydrated in increasing concentrations of ethanol (70 and 100 % for 2 min each) and then were airdried. For using the bright-field microscopy in this SCD assay, the slides were covered horizontally with a mix of Wright’s staining solution for 8 min with continuous airflow. Slides were then washed and allowed to dry. Strong staining was preferred for visualizing the periphery of the dispersed DNA halos. As provided by the manufacturer (Exelixis®, Halosperm®G2) spermatozoa with big and medium halo, while spermatozoa with small, without halo and degenerated were considered to be without and with fragmentation, respectively. The sperm DNA fragmentation index (SDF) was calculated by the form SDF (%) = fragmented + degenerated/total cells counted. For the present study, a minimum of 300 spermatozoa per sample were scored under the 100× objective of the microscope. In order to reduce the bias, two different technicians counted at least 150 spermatozoa each.
Alcohol assessment Statistical analysis “How many drinks of alcohol have you been drinking the previous year until now per day or per week?” was another question concerning alcohol consumption. The
13
Data were normally distributed (one-sample Kolmogorov– Smirnov test). ANOVA was performed for comparison
Arch Gynecol Obstet
between groups followed by the least square differences post hoc testing. Bivariate correlation between variables was tested with Pearson’s correlation. Whenever appropriate, univariate analysis was used. A p value £0.05 was considered statistically significant. Data are expressed as mean ± standard error of the mean (SEM). SPSS v17 statistical package was used to perform the statistical analysis.
Results Table 1 represents the demographic variables, semen parameters, SDF, as well as smoking and alcohol statuses of all men studied. Interestingly, SDF overall inversely correlated with the concentration (r = −0.53, p
Gynecologic Endocrinology and Reproductive Medicine
The impact of cigarette smoking and alcohol consumption on sperm parameters and sperm DNA fragmentation (SDF) measured by Halosperm® G. Anifandis · T. Bounartzi · C. I. Messini · K. Dafopoulos · S. Sotiriou · I. E. Messinis
Received: 20 January 2014 / Accepted: 2 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Both cigarette smoking and alcohol consumption are somehow implicated in sperm function, but the impact of these two lifestyle factors on sperm parameters remains controversial. The present study is focused on the impact of cigarette smoking and alcohol consumption separately and combined on sperm parameters and sperm DNA fragmentation (SDF). Methods The study included 207 consecutive semen samples derived from men who were seeking semen analysis for fertility purposes in our IVF Unit. Results Semen volume, percent of degenerated spermatozoa and SDF were significantly correlated with the various smoking status. The percent of spermatozoa with small halos significantly correlated with the alcohol status. The smoking status of the men was correlated with the alcohol status. Conclusions Cigarette smoking and alcohol consumption separately and combined were found to have deleterious effect on sperm parameters and SDF. It is suggested that both habits may contribute to infertility problems. Keywords Alcohol consumption · Cigarette smoking · DNA fragmentation · Semen quality Introduction Male infertility accounts for approximately 40 % of infertile couples. However, a specific male disorder may not G. Anifandis (*) · T. Bounartzi · C. I. Messini · K. Dafopoulos · S. Sotiriou · I. E. Messinis Department of Obstetrics and Gynaecology, Faculty of Medicine, School of Health Sciences, University of Thessalia, Larisa, Greece e-mail: [email protected]
always be identified and for this reason environmental factors, such as estrogenic pollutants [1], obesity, and lifestyle factors such as cigarette smoking and alcohol intake [2] have drawn much attention. Cigarette smoking and alcohol consumption have been proposed to impact on male fertility and subsequently on reproductive outcome, since the effects that both exert on global and individual health are well known [3]. Nicotine, the main component of cigarettes, has been found to have deleterious effects on sperm parameters [4]. Chronic exposure of spermatozoa on toxic components, such as nicotine or its metabolite cotinine, have been associated with sperm cell death, a situation that is derived after the damage of sperm DNA [5]. When assisted reproduction is used, the outcome is relatively low, because smoking through a molecular mechanism produce spermatozoa with chromosomal abnormalities [6]. Multiple studies have proved that smoking induces alterations in semen including increase in reactive oxygen species (ROS) and decline in antioxidant molecules [7, 8]. It has been reported recently that smoking has a negative effect on sperm parameters, including semen volume, sperm concentration, motility and DNA fragmentation [9–11]. However, other studies failed to find any impact of cigarette smoking on sperm parameters [12, 13]. Several studies focused on the association between alcohol consumption and male fertility. Alcohol intake has been related to reduced sperm parameters including semen volume, sperm count, motility and morphology [14]. Excessive alcohol consumption has been implicated in the alteration of serum hormonal levels, which in turn result in the reduction of seminal plasma fluid [15], underlying a possible inhibitory role of alcohol on gonadotrophin secretion [16]. On the contrary, there are few data that failed to observe any impact of alcohol intake on sperm parameters
13
[17], possibly because most studies are based on the average weekly or monthly alcohol consumption and not on the acute (last 5 days) alcohol intake. Even then Hansen et al. [18] failed to find any significant impact of alcohol on sperm parameters. The impact of smoking and alcohol separately on sperm parameters has been investigated, but with controversial findings. Nevertheless, studies about the effect of both smoking and alcohol consumption are scarce. Moreover, these studies have not reached yet a solid conclusion about the combined impact of both cigarette smoking and alcohol intake and for that reason we undertook this study in order to shed more light relatively on the effect of both factors on sperm parameters and sperm DNA fragmentation (SDF).
Patients and methods Two hundred and seven semen samples obtained by 207 men, for fertility purposes in our Assisted Conception Unit, were prospectively investigated after informed consent and approval of Institutional Review Board. Before semen analysis, a questionnaire was distributed to all men. In the specific brochure, questions concerning their age, height, weight, medical history, smoking habits and alcohol consumption were included. All samples were obtained by masturbation after sexual abstinence of 3–4 days and semen analysis was performed according to 2010 WHO guidelines [19]. In the present study, we evaluated seminal volume, semen concentration, progressive motility (PRM), non-progressive motility (NPR), immotility (IM) and sperm DNA fragmentation index with the Sperm Chromatin Dispersion assay (SCD), known as Halosperm®. Smoking assessment “How many cigarettes have you been smoking (per day) the last year until the current semen sample?” was one of the questions concerning smoking status. The measurement of smoking status was performed according to the type of the pack-year history ranged from 0 to 2.5 pack-year history, which means that a man with a 2.5 pack-year history smokes 2.5 packs of cigarettes per day/per year. The explanatory variable (cigarette smoking) was categorized in three groups: no smokers (n = 98), moderate smokers (0–1 pack-year history, n = 76), heavy smokers (>1 packs-year history, n = 33).
Arch Gynecol Obstet
measurement of alcohol intake was estimated by alcohol units: 1 alcohol unit was considered for 100 ml wine, 200 ml beer, for 30 ml whisky and vodka. The explanatory variable (alcohol intake) was divided into three groups: no alcohol users (n = 112), moderate alcohol users or alcohol users drinking >0 to 7 units/week (n = 25). Assessment of DNA fragmentation Sperm DNA damage was performed with the sperm chromatin dispersion (SCD) assay, known as the Halosperm® G2 kit. Briefly, a maximum of 20 μl of the semen sample was diluted to 20 million/ml in phosphate-buffered saline (PBS). Eppendorf tubes were placed in a water bath at 90– 100 °C for 5 min in order the agarose to be fully melted. After 5 min of incubation, we transfer 50 μl of the diluted semen sample to the 100 μl melted agarose tube and we mix gently with a pipette. We placed immediately 8 μl of the cell suspension onto the sample well and we cover with a coverslip. Slides were then placed on a gold plate in the refrigerator (4 °C) for 5 min in order for the agarose to be solidified with the sperm cells embedded within. After taking out the slides of the fridge, the coverslips were gently removed and the slides were placed horizontally in an elevated position. We then apply with the denaturant agent, making sure the slide is fully covered and we incubate for 7 min. The slides afterwards were horizontally immersed in a lysis buffer and incubated for 20 min. After washing with abundant distilled water for 5 min, the slides were dehydrated in increasing concentrations of ethanol (70 and 100 % for 2 min each) and then were airdried. For using the bright-field microscopy in this SCD assay, the slides were covered horizontally with a mix of Wright’s staining solution for 8 min with continuous airflow. Slides were then washed and allowed to dry. Strong staining was preferred for visualizing the periphery of the dispersed DNA halos. As provided by the manufacturer (Exelixis®, Halosperm®G2) spermatozoa with big and medium halo, while spermatozoa with small, without halo and degenerated were considered to be without and with fragmentation, respectively. The sperm DNA fragmentation index (SDF) was calculated by the form SDF (%) = fragmented + degenerated/total cells counted. For the present study, a minimum of 300 spermatozoa per sample were scored under the 100× objective of the microscope. In order to reduce the bias, two different technicians counted at least 150 spermatozoa each.
Alcohol assessment Statistical analysis “How many drinks of alcohol have you been drinking the previous year until now per day or per week?” was another question concerning alcohol consumption. The
13
Data were normally distributed (one-sample Kolmogorov– Smirnov test). ANOVA was performed for comparison
Arch Gynecol Obstet
between groups followed by the least square differences post hoc testing. Bivariate correlation between variables was tested with Pearson’s correlation. Whenever appropriate, univariate analysis was used. A p value £0.05 was considered statistically significant. Data are expressed as mean ± standard error of the mean (SEM). SPSS v17 statistical package was used to perform the statistical analysis.
Results Table 1 represents the demographic variables, semen parameters, SDF, as well as smoking and alcohol statuses of all men studied. Interestingly, SDF overall inversely correlated with the concentration (r = −0.53, p
