Semen Quality in the General Population of Bulgaria

Background: With the accumulating evidence of semen difference between distinct populations we decided to conduct a population-based prospective study to assess the reproductive capabilities of the Bulgarian males. Objec-tive: To evaluate the semen parameters in men from the general population of Bulgaria. To analyze the effects of BMI (Body mass index) and tobacco smoking on semen characteristics. Methods: 482 males without history of reproductive problems were included for the purpose of this study. The duration of our study was from April to May in 2016 and April in 2017. The volunteers were subdivided based on their smoking status and on their BMI. After semen collection, a conventional semen analysis was carried out manually by trained embryologists in an andrology laboratory according to WHO recommendations. The results were statistically analyzed and presented with their mean values. Result(s): After semen analysis we report a mean of 3.29 ml for semen volume, 40.68 × 10 6 /ml for spermatozoa concentration, 128.38 × 10 6 for total sperm count, 52.54% of total motile spermatozoa in the ejaculate and a mean percentage of morphologically normal sperm standing at 9.6%. We did not find a negative effect of tobacco smoking on semen quality; however higher BMI is associated with lower sperm concentration per ml. Discussion and Conclusion(s): This is the first contemporary study, assessing semen quality in Bulgarians. The results obtained here show the lower quality of semen in Bulgaria, compared to other countries. No statistical differences were found between the semen quality of nonsmokers and smokers. Higher BMI was found to be associated with lower sperm concentration per milliliter.


Introduction
It is a well-known fact that fertility decreases with the increase in age in both men and women. Since the advent of assisted reproductive technology (ART) people are becoming more and more dependent on these techniques in order to have offspring. This is due to the fact that various causes of infertility can be bypassed by the use of ART; however frequently the primary condition remains untreated. While female infertility has drawn more attention, it is proposed that 1 in 20 men are infertile on worldwide scale [1]. Moreover, the cause of infertility in about 20% -40% of infertile couples is attributed to the male partner [2].
This statement calls for more research regarding the possible causes of male infertility and how can we overcome them. From a medical point of view, it is important to reveal the exact cause of infertility as it could be due to some form of cancer, which if left untreated may affect not only patient's fecundity. On a personal level, infertility could cause potential psychological problems.
Semen quality is directly related to men's reproductive capabilities and there is accumulating evidence of the deterioration of semen parameters during the last 40 -50 years [3]. This aggravation is connected to many various factors like smoking, stress, alcohol consumption, age and many others. Environmental toxins and dietary patterns could further worsen the quality of men's semen. Additionally, the higher incidence of testicular dysgenesis syndrome on a worldwide scale also impacts semen parameters negatively. Interestingly, in the literature have been reported differences between the semen parameters of men from different geographical regions [4]. And since this establishment, various studies have been published in order to assess different populations' seminal parameters [5] [6]. Even more interesting is the fact that these differences aren't well defined. Differences in style and quality of life in different countries, geographical peculiarities and many other factors all contribute to these differences. Virtanen et al. conducted a large meta-analysis in 2017 underlining the difference between semen parameters of men from different countries [7]. The study included many countries from all around the world; however there was no available data regarding Bulgaria. Moreover, we could not find any study reporting semen characteristics from the general population in Bulgaria in the last 30 years. Therefore, we decided to conduct a population-based prospective study with the goal of assessing semen quality in Bulgaria and to compare it to other publications on this topic. As a secondary goal we decided to analyze the role of BMI and tobacco smoking on semen parameters due to the fact that there isn't a unified answer on these topics.

Patient Selection
In order to recruit volunteers, we designed a campaign with the goal of assessing male's reproductive health in Bulgaria, based on semen analysis. The study was approved by the ethical committee of Sofia Hospital of Obstetrics, Gynaecology were also questioned if they had consumed alcohol the day prior to semen collection and if they had used any antibiotics the past two weeks.

Sample Collection and Analysis
Semen samples were collected in plastic sterile containers by masturbation after a sexual abstinence period of 2 -7 days. All semen samples were manually evaluated after 30 minutes of liquefaction at room temperature for volume, sperm concentration, morphology and motility while pH and the presence of leukocytes, sperm agglutination and aggregation were also noted, but not used in this study. The semen analysis was performed by trained embryologists in an andrology laboratory. All samples were analyzed within 1 hour after semen collection. The analysis was made according to the 5th WHO guidelines [8]. Semen volume was measured by the aspiration method and the results were presented as milliliters. Motility was expressed as a percentage of normokinetic, hypokinetic and immotile spermatozoa when at least 200 spermatozoa were analyzed.

Distribution of Cases
Based on the results from the semen analysis we further divided the subjects.
The distribution of cases was performed in line with WHO recommendations [8]. Therefore, a patient with a sperm concentration less than 15 million per milliliter was classified as having an Oligozoospermia. Azoospermia was defined as absence of spermatozoa in the ejaculate after centrifugation. Asthenozoospermia included samples with less than 32% normokinetic spermatozoa. Normozoospermia included samples with volume greater than 1.5 ml, sperm concentration per ml greater than 15 million, normokinetic spermatozoa more than 32% and more than 14% of morphologically normal forms. Cases, featuring combined abnormalities were classified by the aforementioned values.

Statistical Analysis
All statistical analysis was performed by GraphPad InStat and GraphPad Prism 6. We calculated the mean, median (5th -95th percentile) and standard deviation for all semen parameters based on the untransformed raw data. Semen parameters are not represented in median, but in mean data for convenience. We used Mann-Whitney U test (two-tailed) when assessing the influence of tobacco smoking on the semen variables. Kruskal-Wallis test was used to compare the semen variables between the different BMI groups. Statistically significant differences were reported if P < 0.05.

Subject Characteristics
The mean age of the 482 patients was 34.38 (SD 6.62). In order to analyze the effects of tobacco smoking and BMI on the semen parameters the participants were subdivided based on their smoking status and BMI. Unfortunately, we did not have complete information for the whole sample size and therefore we ended up with 203 nonsmokers and 73 smokers. In the BMI group we had 276 subjects which were further subdivided in three groups-104 patients with BMI < 24.9; 122 patients with BMI between 25 and 29.9; 50 patients with BMI > 30.

Semen Variables
Semen parameters are presented in Table 1

Tobacco Smoking and Semen Characteristics
Descriptive statistics from patients separated by their smoking status is presented in Table 2. We found a higher number of total sperm concentration and concentration per milliliter in the nonsmokers group compared to the smokers' one. Despite this fact, when comparing the results, we found no statistically significant difference between the two groups (P > 0.05). Other semen characteristics showed very subtle distinction.

BMI and Semen Characteristics
Patients were allocated in three groups according to their BMI. Group 1 included 104 patients with BMI < 24.9; Group 2 -122 patients with BMI between 25 and 29.9; 50 Subjects with BMI > 30 were allocated in Group 3. There were some differences between groups' mean semen volume, motility and morphology ( Table 3). The most substantial differences between groups were seen when comparing sperm concentration per ml and total sperm concentration. It was found that there is a significant statistical difference between sperm concentration per ml between the different BMI groups (P = 0.037). Although the obvious difference in total sperm concentration, statistical significance was not reported. Additionally, we found no significant difference when comparing other semen characteristics (P > 0.05).

Discussion
This is the first contemporary study to assess the quality of semen of healthy implies that sperm morphology is the most common abnormal parameter. The distribution of cases is a scarce statistic and therefore we couldn't find appropriate studies to which we can compare our results. Most studies focus around sperm parameters' raw data without classifying the participants further based on the results. Azoospermia was presented in 1.66% of our study subjects which is lower than the 6.2% reported in Nigeria [11] and 5.30% in France [12]. Regarding other semen abnormalities we didn't find appropriate studies to compare with because those studies that indeed had distribution of cases were using very different patient selection thus making juxtaposing questionable.
With respect to semen parameters again we tried to find appropriate studies for comparison. However, each study has its own methods, statistics and frequently-different patient selection criteria. All of the aforementioned facts make juxtaposing unreliable. Moreover, there are not many studies that evaluate the semen quality in a certain region based on men from the general population. Most studies like those originating from the Baltic countries focus around very young men, neglecting those over 20 years old [13]. Although these studies are very well conducted and follow almost the same pattern, they give information regarding the younger part of the population. Their study model has been used in other countries too due to its fine structure [5] [6]. These studies, based on a same model are easily comparable and they underline the difference in semen parameters between men from different countries. Other studies focus on patients with infertility or those that are proven fertile [4].
In Table 4   this was our selection criteria. Although there are differences in the methods between the studies, the results we obtained here are some of the lowest reported which questions the lifestyle and the health status of Bulgarian males. When comparing mean sperm concentration per ml it was found that from the data we had obtained only in Poland and Ukraine had been reported lower mean data [14]. Data from Germany (Paasch et al., 2008), China [15] [16], Faroe Islands [17], Spain [18], Turkey [19], Sweden [20] and Denmark [21] were all higher when comparing both types of sperm concentration to our findings. Some of the studies showed significantly higher parameters compared to our findings. Mean semen volume varies between studies, ranging from 2.5 ml to 4.1 ml (Table 4) while in our study we reported a mean of 3.29 ml. Many investigators do not include normokinetic and hypokinetic spermatozoa, but prefer to measure the total motility in their studies [16] [17]. Our mean data of this parameter was 52.5% which was lower than the values reported from Faroe Islands, China, Germany, Murcia, Turkey and Denmark however it was higher than the Barcelona study (Table 4). From the studies that indeed included normokinetic parameter, our mean value of 44.7% was lower than all reported with the exception of the study of Wdowiak [14]. In terms of morphology, we report 9.6% normal forms which is an average value compared to the studies. We must note that in all studies we had included in our comparison  [22]. For example, DNA repair mechanisms failure is connected to increased likelihood of carcinogenesis [23].
Along with the evaluation of semen quality in Bulgarians we also examined the effect of tobacco and BMI on semen quality. There are numerous studies on this subject with mixed conclusions as some say that smoking does indeed worsen semen characteristics [24], but others say it does not [16]. According to our results, smoking does not have statistically significant effect on semen quality, although these results should be interpreted with caution due to the relatively small sample size.
The effect of BMI on semen quality is well studied although the results are not consistent. Keskin et al. in 2016 reported that BMI is not connected to semen parameters [25]. On the contrary, Andersen et al. in 2015 stated that BMI has a negative effect on semen quality, especially in males with BMI over 35 [26]. Our results imply that higher BMI is correlated with lower semen concentration per ml. Other parameters did not show statistical difference despite the obvious higher parameters in Group 1 (Table 3).
We believe that due to our inclusion criteria, the participants in this study are representatives of the general population of Bulgaria; however our results should be confirmed or questioned by other larger studies in the future in order to definitely confirm this statement.
As a possible drawback to our study is the lack of external quality control, used in other studies [6]. Other possible causes of limitations could be that only one sample was analyzed per subject. Another source of possible drawbacks is connected to the reluctancy of some participants to give more information about their lifestyle and fertility problems; however this problem can't be avoided.
Hopefully, the results from this study would underline the difference between semen characteristics among different geographical regions and urge for more research on this topic.

Conclusion
This is the first study, reporting semen quality in Bulgaria. Mean semen characteristics were lower than most of the data reported in other studies. Adverse semen parameters in Bulgaria are probably connected to the lifestyle of men and to the amount of stress they cope with. Moreover, the quality of life in Bulgaria is not that high when compared to other European countries. Furthermore, here we confirm that there are indeed differences in semen quality between different regions. We did not find a negative effect of tobacco smoking on semen quality.
Higher BMI is connected to lower sperm concentration per ml.