The study population consisted of men, who were referred to the Reproductive Medicine Center of Shanxi Women and Infants Hospital (Taiyuan, China) between January 2013 and January 2015. All subjects were of the Han population from Shanxi Province in north China. The study was approved by the ethics committee of the Shanxi Women and Infants Hospital (Taiyuan, China) and the individuals in the relationships provided consent.

The inclusion criteria were as follows: Being the male partner of an infertile couple for a duration of at least 1 year, having regular intercourse, and seeking infertility treatment at the Reproductive Medicine Center, Shanxi Women and Infants Hospital over the study period. A careful history was obtained from each subject to exclude systemic diseases and assess alcohol assumption; careful physical examination was performed, with measurement of testicular size to exclude abnormalities of the external genitalia and cryptorchidism; ultrasonographic examination was performed to exclude varicoceles; microbiological examination and spermioculture were performed to exclude infections; an immunobead binding test was performed to exclude the presence of anti-sperm antibodies; karyotyping was used to exclude any chromosomal abnormality; and genetic examination was performed to exclude Y chromosome microdeletions and cystic fibrosis gene mutations.

A brief medical history was obtained, primarily by informal interview with the patient, or from the patient's clinical notes or a self-reported questionnaire. According to the standardization of the World Health Organization (WHO) on smoking and associated literature (18–20), 1,218 men were divided into a smoking group (920 cases) and non-smoking group (298 cases). The smoking group was grouped into three groups, according to daily cigarette consumption, as follows: Mild smoking group (256 cases; ≤9 cigarettes/day), moderate smoking group (365 cases; 10–19 cigarettes/day), heavy smoking group (299 cases; ≥20 cigarettes/day). The smoking group was grouped into another three groups, according to the number of years of smoking, as follows: Short-term smoker group (268 cases; ≤5 years), medium-term smoker group (282 cases; 5–10 years), long-term smoker group (370 cases; ≥10 years).

A semen sample was obtained from all subjects via masturbation following 2–7 days of abstinence for routine sperm counts, according to the WHO (2010) criteria (sperm concentration, motility, morphology and viability) (21). Briefly, the ejaculate volumes were estimated by specimen weight, assuming a semen density of 1.0 g/ml. Sperm concentration, motility and viability were detected using a sperm class analyzer (CASA system; Microoptic S.L., Barcelona, Spain). Sperm motility was also analyzed using the WHO (2010) criteria of progressive motility, non-progressive motility and immotility. In this analysis, the percentage of motile sperm refers to the percentage of sperm with any flagellar movement, whether twitching or progressive. A single technician assessed sperm morphology using the strict methods recommended by the WHO (2010). In addition to the primary measurements of semen quality (sperm concentration, volume and percentage of motile sperm), the CASA system was used to analyze the total sperm count and total motile sperm count.

Sperm viability was assessed within 30 min of ejaculation. Analysis was performed using eosin Y staining (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), for which 1 g of eosin was dissolved with 1 g fresh sperm. The percentage of viable sperm, indicated by an unstained sperm head, and non-viable sperm, indicated by staining of the sperm head, was assessed by counting a minimum of 200 spermatozoa. Replicate counts of 200 sperm on each of two slides were performed using a using a CX31 microscope (Olympus Corporation, Tokyo, Japan), which were then repeated if >5% difference was found (4).

The spermatozoa in each group were analyzed for acrosin activity using a Human Spermatozoa Acrosin Activity Quantitative Assay kit (Huakang Biotech, Shenzhen, China), according to manufacturer's protocol. At 24°C, the quantity of substrate hydrolyzing 1.0 µmol BAPNA/min was defined as 1IU acrosin activity, and determined based on optical density (OD) values: Acrosin activity (µIU/10⁶ spermatozoa) = {[(sample tube OD value-control tube OD value) × 2]/(495 × 7.5)} × 10⁶. OD values were assessed on a DNM-9602 ultraviolet spectrophotometer (Beijing Perlong New Technology Co., Ltd., Beijing, China).

The seminal fluid in each group was analyzed for zinc using a Seminal Plasma Zinc Quantitative Assay kit (Huakang Biotech), according to the manufacturer's protocol. The seminal fluid (1 ml) was centrifuged for 5 min at 1,000 × g at 5 min. The supernatant was transferred into a test tube for use in seminal plasma analysis. The sediment was washed with 1 ml physiological saline solution, mixed on an XH-B vortex-type mixer (Jiangsu Kangjian Medical Apparatus Co., Ltd., Taizhou, China) for 30 sec, and centrifuged again, as previously. The supernatant was discarded and the sediment was used for zinc determination, rather than using 200 µl of liquid sample. The absorbance of the solutions was measured at 490 nm on aHR801 microplate reader (Shenzhen Highcreation Technology Co., Ltd., Shenzhen, China). The concentration in the sample was determined using the following formula: Seminal plasma zinc (µmol) = zinc concentration (mmol/l) × semen volume (ml).

The analysis of DNA fragmentation was performed in fresh semen using fluorescence staining with a kit supplied by (Huakang Biotech), based on the fluorescence emission from individual sperm, which were stained with acridine orange (AO). AO molecules are intercalated into double-stranded DNA, and green fluorescence is emitted from the sperm nuclei. The DNA in sperm with immature nuclei are readily denatured into single strands and, following AO molecule aggregation in the nuclei, the color of the fluorescence becomes orange-red. The cell suspension was pipetted onto a glass slide and observed under a BX51 fluorescence microscope (Olympus Corporation) with a 480–490 nm filter. The percentages of green (normal DNA integrity) and orange-red (abnormal DNA integrity) spermatozoa in each sample of 200 spermatozoa were calculated by the same examiner. The integrity of sperm nuclear DNA was considered abnormal when the percentage of denaturation (orange-red spermatozoa on AO staining) was >34%.

RT-qPCR analysis was used to assess the transcriptional expression of tumor-associated genes, including Chk1. Total RNAs were extracted from the spermatozoa using a Total RNA Purification kit (Nanjing KeyGen Biotech Co., Ltd.), the concentration of the RNA was determined using a NanoDrop 1000 (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and reverse transcribed using an Transcriptor First Strand cDNA synthesis kit (Nanjing KeyGen Biotech Co., Ltd.). The reaction was performed in the following conditions: 30°C for 10 min; 42°C for 30 min; 99°C for 5 min; and 5°C for 5 min. qPCR was conducted in a CFX-96 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a One Step SYBR PrimeScript RT-PCR kit (Takara Bio., Inc.), as described. The specific primers (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were as follows: Chk1, forward 5′-ATATGAAGCGTGCCGTAGACT-3′ and reverse 5′-TGCCTATGTCTGGCTCTATTCTG-3′; GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. qPCR was performed as follows: Initial denaturation at 98°C for 5 min; 40 cycles of 95°C for 10 sec, and 72°C for 15 sec. The 2^−ΔΔCq method was used for quantification, calculated according to the manufacturer's protocol, with results expressed as the mean ± standard deviation.

Spermatozoa were prepared from each group for western blot analysis to determine the expression level of the tumor-associated protein, Chk1. The seminal fluid (1 ml) was centrifuged for 5 min at 1,000 × g. The precipitate was washed with 1 ml physiological saline solution, mixed on a vortex-type mixer for 30 sec, and centrifuged again, as above. For analysis of cellular protein levels, spermatozoa cells were rinsed twice with ice-cold phosphate-buffered saline and then lysed in ice-cold lysis buffer [containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ETDA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin) for 30 min. Cell lysates were centrifuged at 13,000 × g for 10 min at 4℃ and the protein concentration was determined by the Bradford assay. Proteins (20 µg) were loaded onto 8% SDS-PAGE gels for electrophoresis and then transferred onto nitrocellulose membranes. The transferred nitrocellulose membranes were blocked with 5% dried skim milk for 1 h at room temperature, then incubated with mouse anti-Chk1 monoclonal antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; cat. no. sc-377231) at 4°C for 12 h. The membranes were then exposed to goat anti-mouse or rabbit secondary antibody (1:200; Zhongshan Bioengineering, Beijing, China; cat. no. PV9005) in blocking buffer for 1 h at room temperature. The bands were demonstrated by enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Inc.) for 1 min and analyzed using Image-Pro Plus 5.1 software (Media Cybernetics, Inc., Rockville, MD, USA).

All data were analyzed using SPSS 17.0 (SPSS, Inc. Chicago, IL, USA). Normally distributed data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. To assess the normality of the distribution, a Shapiro-Wilk test was performed. One-way analysis of variance was used for comparison among multiple groups if the variance was homogeneous, whereas non-normally distributed variables were analyzed using a Mann-Whitney U test or Kruskal-Wallis variance analysis, as appropriate.

As shown in Tables I and II, routine semen parameters in the smoking groups were compared with those of the non-smoking group. No significant differences in semen volume, sperm concentration or sperm count were found between the smoking and non-smoking groups (P>0.05). However, progressive motility in the smoking group was significantly decreased (P<0.05), compared with the non-smoking group. No significant changes in routine semen parameters were observed in the mild smoking group (P>0.05), whereas the moderate and heavy smoking groups had significantly decreased progressive motility (P<0.05). No significant differences were found in the routine semen parameters in the short-term smoking group, compared with the non-smoking group (P>0.05), whereas the medium-term smoking group had significantly decreased progressive motility (P<0.05), and the long-term smoking group had decreased semen concentration, sperm count and progressive motility (P<0.05).

As shown in Tables III and IV, the sperm morphology in the smoking groups was compared with the non-smoking group. No significant differences were found in the sperm morphology in the normal sperm count and abnormal head, body and tail counts between the smoking and non-smoking groups (P>0.05). Compared with the non-smoking group, no significant changes were found in the sperm morphology between the mild smoking group and moderate smoking group (P>0.05), nor were there significant differences between the short-term smoking group and medium-term smoking group (P>0.05). However, the abnormal head rate in the heavy smoking group and long-term smoking group were significantly higher, compared with those in the non-smoking group (P<0.05).

Sperm viability was analyzed using eosin Y staining. As shown in Fig. 1A, the viable sperm, in which sperm heads remained unstained, and non-viable sperm, in which sperm heads were stained, were examined using a CX31 microscope (Olympus Corporation). Compared with the non-smoking group, the smoking group had significantly decreased sperm viability (Fig. 1B; P<0.05).

As shown in Fig. 2A, the seminal plasma zinc concentration decreased significantly in the smoking group, compared with the non-smoking group (P<0.05). Similar results were obtained for spermatozoa acrosin activity between the smoking group and non-smoking group (Fig. 2B; P<0.05). DNA fragmentation rates were analyzed using AO staining. As shown in Fig. 2C, the green (normal DNA integrity) and orange-red (abnormal DNA integrity) spermatozoa were examined using a BX51 fluorescence microscope (Olympus Corporation) with a 480–490 nm filter. Compared with the non-smoking group, the smoking group exhibited a significantly higher DNA fragmentation rate (Fig. 2D; P<0.05).

Analyses using RT-qPCR (Fig. 3A) and western blot analyses (Fig. 3B) demonstrated that the expression of Chk1 was significantly decreased in the smoking group, compared with the non-smoking group (P<0.05).

The present study found a non-linear association between the relative mRNA expression of Chk1, and the progressive motility and sperm concentration (P<0.05). However, DNA fragmentation rates were inversely association (P<0.05) with progressive motility and sperm concentration (Table V).