The reference values for semen parameters of 1213 fertile men in Guangdong Province in China.

Semen samples were collected from 1213 fertile men whose partners had a time-to-pregnancy (TTP) ≤12 months in Guangdong Province in Southern China, and semen parameters including semen volume, sperm concentration, total counts, motility, and morphology were evaluated according to the World Health Organization (WHO) 2010 guideline. All semen parameters analyzed were normal in ~62.2% of the total samples, whereas ~37.8% showed at least one of the semen parameters below normal threshold values. The fifth centiles (with 95% confidence intervals) were 1.3 (1.2-1.5) ml for semen volume, 20 × 10 6 (18×10 6 -20×10 6 ) ml-1 for sperm concentration, 40 × 10 6 (38×10 6 -44×10 6 ) per ejaculate for total sperm counts, 48% (47%-53%) for vitality, 39% (36%-43%) for total motility, 25% (23%-27%) for sperm progressive motility, 5.0% (4%-5%) for normal morphology. The pH values ranged from 7.2 to 8.0 with the mean ± standard deviation at 7.32 ± 0.17. No effects of age and body mass index were found on semen parameters. Occupation, smoking and alcohol abuse, varicocele appeared to decrease semen quality. Sperm concentration, but not sperm morphology, is positively correlated with TTP, whereas vitality is negatively correlated with TTP. Our study provides the latest reference values for the semen parameters of Chinese fertile men in Guangdong Province, which are close to those described in the new WHO guidelines (5 th Edition).

INTRODUCTION

Semen quality has been commonly regarded as a measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology, and pregnancy risk assessments.1 Since the publication of a meta-analysis showing a major decline in sperm counts over a period of 50 years after the second world war,2 many retrospective, comparative studies on semen quality around the world have been published over the past decade. Several reports suggested a decline,345678 whereas others showed no significant changes in semen quality in men.91011121314 Similar debatable data on the semen quality in China have also been published; by reviewing 115 reports published between 1985 and 2009, Huang et al.14 analyzed the data from 23 126 healthy Chinese subjects and demonstrated a possible decline in sperm concentration. In contrast, Wen et al.15 reported no evidence of decline in sperm concentration during a period of 18 years in Guangdong Province in Southern China.

Semen quality has been considered as one of the most sensitive indicators of the adverse effects of environmental pollution.16 In addition to physical environments, semen quality is also affected by other factors, such as age,1718 occupation,19 cigarette smoking,2021 and lifestyle.2223 Most of the previous studies used less defined semen samples from laboratories in fertility clinics, whereas two recent studies from China investigated well-defined groups representative of the population of healthy young men.2425 Other deficiencies of previous studies, especially those published in the earlier years,262728 include the lack of comparable laboratory techniques and inappropriate statistical analyses. The present study was aimed at a comprehensive assessment of the current status of semen parameters in Chinese fertile men whose partners had time-to-pregnancy (TTP) ≤12 months in Guangdong area according to the strict guideline of World Health Organization (WHO) laboratory manual for the examination and processing of human semen (2010, 5th Edition).

MATERIALS AND METHODS

Subjects

Husbands of pregnant women who had TTP ≤ 12 months from Heyuan, Jiangmen, Yangjiang, Zhanjiang, and Qingyuan areas in Guangdong province were invited to participate in our study from October 2010 to September 2012 by the local Family Planning Network. The total of 1258 fertile men were invited to participate and 1213 (96.4%) agreed. The eligibility criteria for the male participants were as follows: age 20–45 years at the time of invitation, residence of the local area near the hospital where he was recruited. In addition, the woman's current pregnancy had to be achieved by normal sexual intercourse rather than fertility treatment. Subjects with the following conditions were excluded: epididymitis, cryptorchidism, orchitis, genital tract surgery, chemotherapy, radiotherapy or chronic illness, previous treatment for infertility or reduced fertility, and unwanted pregnancy or prolonged TTP.

Questionnaires

Both the men and their pregnant wives were asked to complete a questionnaire. The questionnaire included detailed information on demography, education, lifestyle, occupational exposure, reproductive history, consumption of tobacco and alcohol, and previous or current diseases. The participants were informed of the purpose of the study, and possible benefits and risks of participating in the study. All participants were asked to sign an informed consent form if they agreed to take part in this study. The protocol for the use of human subjects was approved by the Human Subject Ethics Committee of the Research Institute of Family Planning of Guangdong Province.

Physical examination

Physical examinations of all subjects were performed by the same two experienced andrologists. The results of examinations were recorded in a standard form. Secondary sexual characteristics and the possible presence of a varicocele, a hydrocele, the location of the testis in the scrotum, and the consistency of the testis and epididymis were examined to exclude the subjects with reproductive or urological diseases. Weight (kg) and height (cm) were measured using the same calibrated instrument in each center.

Semen samples

All subjects were asked to abstain from ejaculation for a period of 2–7 days. The period of ejaculation abstinence was calculated as the time between the current and previous ejaculations as reported by the subjects. The exact duration (in days) of abstinence was documented for each participant. The participants collected the ejaculates by masturbation at the local Family Planning Institutions into a sterile plastic container (MaleFree™SCD-II, BRED Life Science, Shenzhen, China) and immediately delivered the sample to a laboratory in the same building. The semen samples were marked with an anonymous serial number, and were then incubated in a water-bath at 37°C until analysis. The semen volume was measured after samples were liquefied. All samples were analyzed within 60 min after collection.

Sperm motility was analyzed using a computer-assisted semen analysis (CASA) system (SCA2000, Microptic, Barcelona, Spain). According to the WHO reference, percent of motile sperm was scored using category A (rapid progressive motility [PR]), category B (slow PR), category C (nonprogressive motility [NP]), and category D (immotility). Two types of motility were evaluated in the present study: PR and NP. Other CASA parameters analyzed included curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity, beat cross frequency, and amplitude of lateral head displacement. The calculated parameters were linearity (LIN = VSL/VCL × 100) and straightness (STR = VSL/VCL). To avoid using CASA parameters that reflect different aspects of the same movement as described in previous studies,2930 we selected VSL (for progression), VCL (for vigor), and LIN (for swimming pattern) for statistical analyses.

For morphological evaluation, semen smears were fixed with a mixture of absolute alcohol and acetone (at a ratio of 2:1), followed by staining using the modified Shorr solution. Assessment of sperm morphology was conducted according to the criteria published by the WHO, 2010, the 5th edition.

Quality control of semen analysis

To minimize variations in the assessment of sperm characteristics, samples from all five centers were analyzed by the same two well-trained technicians using the same instruments and methods. One technician evaluated appearance, viscosity, liquefaction time, semen volume, while the other measured sperm concentration, motility, and morphology. The two technicians participated in the continuous quality control system, an external quality control system established based on the WHO guidelines, supervised by the Quality Control Center of Guangdong Province. Semen quality was evaluated based on the recommendations by the WHO, 2010.

Statistical analyses

Because semen parameters follow markedly nonnormal distributions, unadjusted percentiles, and medians were calculated for the semen parameters. Percentages coincident with the criteria of WHO (2010) were also calculated. The data were also summarized using median, 5th percentiles, and were stratified by age, body mass index (BMI), occupation and lifestyle. Kruskal–Wallis analysis of variance, a nonparametric test, was used to compare medians between groups. A step-wise multiple regression analysis was used to determine which variables are the strongest independent correlates of TTP. A generalized linear model was used to examine the independent effect of risk factors on semen parameters. A full model that included all risk factors examined in the final regression was used. Independent variables entered into the regression model as dummy variables were as follows: (i) age: ≥35, 30–35, 25–30, and <25 as reference; (ii) BMI: >25.0, <18.5, and 18.5–25.0 as reference; (iii) occupation: others, farmers, and workers as reference; (iv) smoking: yes and no as reference; (v) alcohol consumption: yes and no as reference; (vi) urogenital disease history: yes and no as reference; (vii) varicocele: yes and no as reference. The level of significance was established at 0.05. All semen parameters were log-transformed to improve the normality as dependent variables in the generalized linear models.

RESULTS

Subject characteristics

The mean age of the participants was 31.6 ± 5.2 years (range: 20–45 years). The demographic characteristics of the participants are presented in .

Semen analysis

All semen samples were gray or grayish yellow in color. The liquefaction time was within 60 min. The thread length was no more than 2 cm. the pH values ranged from 7.2 to 8.0, and the mean and standard deviation was 7.32 ± 0.17. All samples were analyzed within 60 min after collection. shows semen characteristics of the 1213 subjects. The normal sperm morphology and sperm concentration were within high-normal values (97.5% and 97.4%, respectively) according to the WHO criteria. Of the 1213 semen samples evaluated, 62.2% (755/1213) displayed normal semen parameters according to the WHO criteria (WHO, 2010), but 37.8% showed below normal threshold values in at least one of the semen parameters, including semen volume, sperm concentration, total count, vitality, PR, and normal morphology.

Risk factors for the semen quality of fertile men

The semen samples were grouped based on age, BMI, occupation, smoking, alcohol consumption, urogenital disease history, and varicocele. The semen parameters were correlated with these variables using step-wise multiple regression analyses. shows adjusted regression coefficients and 95% confidence interval for all possible risk factors in relation to semen parameters. Ages and BMI appeared to have no effects on the semen quality, whereas occupations seemed to affect sperm concentration, total sperm counts, vitality and percentage of forward progression sperm. In addition, smoking had effects on sperm concentration and total sperm counts, and alcohol intake could impact sperm vitality and percentage of forward progression sperm. Interestingly, history of urogenital diseases did not seem to affect the semen quality except that varicocele had a negative impact on sperm morphology.

Summary of time-to-pregnancy

The minimum and maximum TTP of 1213 fertile men whose partners achieved pregnancy within 12 months of unprotected sexual intercourse was 0 and 12 months, respectively. Distribution of the TTP frequency among 1213 women whose male partners participated in this study is shown in . More than 50% couples achieved pregnancy within 6 months. The step-wise multiple regression analyses were used to determine which variables are the strongest independent correlates of TTP. Statistical analyses showed that sperm concentration was positively correlated to TTP (t = −4.801, P < 0.001), whereas vitality was negatively correlated to TTP (t = 3.954, P < 0.001).

The time-to-pregnancy frequency distribution of 1213 women whose male partners participated in this study. The number in the bar represent the number of couples establish pregnancy at this month; the percentage above the bar represent the percentage of couples establish pregnancy at this month. More than 50% couples achieved pregnancy within 6 months.

DISCUSSION

Our data represent the current semen parameters of Chinese fertile men whose partners had TTP ≤ 12 months in Guangdong area. Subjects in the previous reports on semen quality in China were only defined as healthy people or sperm donors, with no or little information on TTP of their female partners.242531 Subjects in our study were all whose partners achieving pregnancy within 12 months. Moreover, our results are not in complete agreement with those reported previously in several large studies in Chinese men.242531323334 As shown in , the mean values of semen volume and concentration in our study (3.0 ml and 80 × 106 ml−1, respectively) are in agreement with those of others (2.5–3.0 ml and 55.9 × 106–99.2 × 106 ml−1, respectively), whereas the mean values of total sperm counts in our study (238 × 106) are markedly higher than those in other studies, ranging from 133.6 × 106 to 203.2 × 106. The mean values of total motility in our study (68.0%) are similar to the other reports (47.7%–77.2%). Compared semen parameters reported here with earlier studies of American (USA) and European (France, Denmark, Finland, Estonia and Norway) men,35363738 the mean semen volume in Chinese men was lower by 0.6–1.4 ml. The mean sperm concentration (80.0 × 106 ml−1) in Chinese fertile men is higher than that in young American and Nordic-Baltic men (range: 54.3 × 106–75.5 × 106 ml−1), but lower than that of French men (95 × 106 ml−1). The mean total sperm counts (238 × 106) in our study was higher than was recorded for men in the USA (range: 113.0 × 106–149.7 × 106), but was markedly lower than that of French men (337 × 106). For the mean total motility, our study indicated that motility in Chinese men (68.0%) was similar to that of European men (range: 64%–73%), but was higher than that in the USA (range: 48.2%–56.4%).

Discrepancies among these studies could result from many factors, such as demographic characteristics, region, lifestyle, environment, and methodology used for semen analyses. For example, semen analysis is a rather subjective technique, and is associated with inter-laboratory variations,3637 making it difficult to compare assessments performed by different laboratories. We also cannot exclude geographic variations in semen quality, since several studies have shown apparent geographic variations after adjusting for possible confounding factors. Together, we speculate that these variations can result from factors including lifestyle, environmental exposure and genetic variations, or a combination of all those factors.

A lack of significant effect of age on semen parameters in this study can well be due to the narrow age range (20–45 years old) of participants because men of this age group are usually at the peak of reproductive capability. Recent studies suggest that increased age is associated with a decrease not only in semen volume, but also in the percentage of sperm morphology and motility.439 Other studies have shown that there is no correlation between sperm concentration and age.4041 Discrepancies among these studies may be due to the different ages of men examined, or due to other confounding factors.

Several studies have demonstrated that BMI could be associated with semen quality. Jensen et al.38 found a significant association between sperm counts and BMI; both overweight and overly slim men had lower sperm concentrations and lower total sperm counts compared to men with ideal body weights (BMI between 20 and 25 kg m−2). Results reported by Kort et al.42 reveal a significant, negative relationship between BMI and the total number of normal-motile sperm; men presenting with a BMI > 25 kg m−2 have fewer chromatin-intact normal-motile sperm per ejaculate. In our study, BMI do not appear to have an effect on semen quality, which is consistent with the results of the Chinese population reported by Gao et al.25

Our study also suggests that occupation might affect semen quality because semen quality of factory workers is significantly lower than that of farmers and others. We speculate that the differences may be attributable, at least in part, to lifestyle and environmental exposures. It is well-known that long-term chronic alcohol abuse causes erectile dysfunction, reduced libido, and gynecomastia. One mechanism of these effects lies in the reduction in serum testosterone levels due to decreased testicular production and increased metabolic clearance in liver. In addition, the oxidation of alcohol competes with testicular production of testosterone. These mechanisms lead to subsequent decrease in semen volume and sperm concentration, which is consistent with our observation that alcohol intake negatively affects semen quality. Another factor appears to be the elevation of serum estrogen levels due to increased peripheral conversion of testosterone to estrogen through enhanced activity of aromatase, which is present in both liver and peripheral adipose cells. It has reported that progressive deterioration in semen quality correlates with increasing quantity of alcohol intakes and cigarettes smoking,43 and smoking has a significant negative impact on sperm production, motility, and morphology.44 Although the underlying mechanism remains unknown, our study further supports those earlier reports, indicating that alcohol abuse and smoking lower the semen quality mainly by reducing the total sperm counts.

Randomized, controlled trials, and prospective studies that evaluate semen parameters before and after varicocelectomy clearly demonstrate that varicocele repair is associated with a significant improvement in sperm concentration, motility, and normal morphology.45 Our study suggests that normal fertile men with varicocele disease can also affect the semen quality, especially the percentage of normal sperm morphology.

Time-to-pregnancy data have been widely adopted to reflect the fecundity of a couple because these data correlate with sperm quality and quantity as well as sexual activity. However, semen studies often have much lower participation rates than those TTP studies, and TTP studies should, therefore, remain in the toolbox for studies of male fecundity.46 The TTP mean value in our study is relatively higher compared to other published data.4748 The negative association between sperm vitality and TTP (t = 3.954, P < 0.001) is easy to understand because poor vitality leads to prolonged TTP. However, the positive correlation between sperm concentration and TTP (t = −4.801, P < 0.001) is counterintuitive and puzzling. One possible explanation might be that overcrowding may interfere with sperm swimming and/or the fertilization process. The lack of correlation between sperm morphology and TTP may not be meaningful because the new WHO criteria used for assessing sperm morphology are too loose, e.g. with a threshold as low as 4%–5%, the majority of men are considered to have normal sperm morphology.

CONCLUSION

This report presents a large study on semen quality in the fertility-proven Chinese men whose partners had TTP ≤ 12 months. Our study revealed that a substantial proportion of Chinese fertile males (~37.8%) had at least one of the semen parameters, including semen volume, sperm concentration, total counts, vitality, PR, and normal morphology, below the normal range. For the fifth centiles of semen parameters, our results are similar to the normal threshold values of the WHO guidelines (5th Edition). We did not find any significant associations between the poor semen quality and age or BMI, but did note that occupation, smoking, alcohol abuse, and varicocele may be important factors that influence semen quality. Sperm concentration positively correlates to TTP, whereas vitality is negatively associated with TTP. This study provides important basic data on semen quality of fertile men in Guangdong area, and the large population dataset presented will be useful for comparison of semen quality in different regions of China.

AUTHOR CONTRIBUTIONS

YGT designed the study, drafted the manuscript, and coordinated and participated in every part of the study. LXT participated in the design of the study and drafting of the manuscript. QLW participated in the design and statistical analyses. GS participated in the design of the study. YJJ performed the semen analyses. SMD analyzed the semen quality and interpreted the data. FJ performed semen analyses and interpreted the data. WBQ helped write and revise the manuscript and supplied important comments. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Table 1

Characteristics of participants

Table 2

Summary of semen parameters

Table 3

Effects of potential risk factors on semen parameters of fertile mena

Table 4

Summary of semen parameters of different regions in China