Determination of Trace Metals in Human Seminal Plasma Using Inductively Coupled Plasma Mass Spectrometry and Multivariate Statistical Analyses for Sperm Parameters

We have simultaneously determined the presence of 30 different trace metals in human seminal plasma by Inductively Coupled Plasma Mass Spectrometry (ICP−MS). To date, high concentrations of metals (e.g. Zn, Cu, Mg and Fe) in seminal plasma have been sporadically reported and only few reports have discussed the relationships between trace metals and human semen profiles. Ninety-four seminal plasma specimens were first classified in three groups (normozoospermia, oligozoospermia, and azoospermia) according to the WHO guideline for sperm concentration. No significant differences were found among the three groups for all the metals examined. The concentration of metals in the specimens were then compared with sperm concentration, motility, morphology, and volume using multivariate regression analysis, principal component analysis (PCA) and cluster analysis. The most cumulative contribution ratio and grouping were extracted for profile analysis. Zn showed a positive correlation with motility and morphology (r = 0.3142 and 0.6205), whereas trace metals such as Se and Tl showed positive correlations with all sperm properties. Heavy metals such as Cu, Cd, and Cr also showed high positive or negative correlations with sperm properties. In summary, this study is the first to report the simultaneous determination of metals (in addition to the commonly reported Zn), including trace and heavy metals, in human seminal plasma as analyzed by multivariate regression analysis. Journal of Molecular Biomarkers & Diagnosis J o u r n a l o f M ole cul ar iorkers & iag n o s i s


Introduction
The general population is usually exposed involuntarily to trace metals through food and water intake or contact with soil, dust, or air. There is growing concern for adverse health effects associated with low-level exposures encountered in the environment. Cd, Pb, and Hg are recognized as hazardous metals that the general population is most exposed [1]. The acceptable daily intake (ADI) and permissible exposure limits (PEL) of trace metals have been determined for the whole human body. Mammalian testes produce large quantities of sperm daily, thus it is one of organs most sensitive to cytotoxic agents such as anti-cancer drugs [2,3]. Metals may have adverse consequences for male reproductive health, especially spermatogenesis, even at relatively low levels. Cr, Cu, Mn, Mo, Se, and Zn, are essential for health maintenance but may be hazardous above allowable limits [4]. Cr, Mn, and Cu, which act as cofactors for a variety of important enzymatic reactions, have been associated with reduced semen quality in rodents and in humans [5,6]. Mo is also an important cofactor for a limited number of enzymes and has demonstrated reproductive toxicity in animal studies [7]. However, low doses of metals such as Cu, Se, and Zn may have protective effects on male reproductive outcomes [8], and may assist in counteracting the effects of Cd, Pb, or other metals that are hazardous to health [9]. To date, human data on non-occupational exposure to these metals have been limited, lacking, or inconsistent across studies, and those for male reproductive functions are more obscure.
The concentrations of metals in human seminal plasma are typically determined by chelatometric titration [10,11] and absorption spectroscopy [12,13]. These popular methods have been used to determine the concentrations of individual metals in prepared sample solutions. In contrast, inductively coupled plasma mass spectrometry (ICP−MS) can be used to simultaneously determine over 30 different metals in the same sample preparation. This is due to its high sensitivity, which is over 1000 times that achieved by chelatometric titration [10,11] and atomic absorption spectroscopy (AA) [12,13].
To evaluate the relationship between metal exposures at environmental levels and male reproductive function, we measured 30 well-studied rich metals and trace metals in human seminal plasma. This was achieved using ICP−MS and compared with semen qualities such as sperm concentration, motility, head morphology, and volume of ejaculate. The metals may affect semen quality either individually or through positive/negative synergetic actions by multiple metals. Most reports have analyzed relationships between one type of metal and the two groups -normozoospermia or abnormalspermia [5][6][7][8], or the WHO classified groups -normozoospermia, oligozoospermia, and azoospermia -by Student's t-test, the Kruskal-Wallis test, and ANOVA [3][4][5][6][7][8][9][10]. Interestingly, various results were observed, including a significant difference among the WHO groups [4] or no significant difference among the WHO groups [9]. Therefore, data was analyzed not only by regression analysis but also by multivariate analyses (principal component analysis: PCA and cluster analysis) for detailed diagnosis of the relationships between the 28 metals and sperm parameters.

Metal concentrations in human seminal plasma
Most reports have analyzed relationships between one type of metal and two groups by semen quality; for example, Meeker et al. [1] classified normozoospermia and sperm morphology (50% normal morphology). The statistical significance or lack thereof was discussed in relation to a single metal and sperm parameters [1,[3][4][5][6][7][8]. Some previous reports have cited that heavy metals such as Cd, Hg, and Pb have hazardous impacts on sperm properties (concentration, motility, morphology [1], and cause DNA damage [1,2]). Zn has been well studied because of its significant role in maintaining male reproductive functions [5], particularly sperm quality [9].
Male infertility treatment was provided for three groups of patients classified in accordance to WHO guidelines [14] at the clinical site. Therefore, it was thought that we should analyze the relationship between metal and sperm properties in the three groups. Most studies have analyzed single metals by titration [10,11] and AA [12,13]. Instead, we planned to simultaneously analyze a number of different metals in seminal plasma for a comprehensive study of the relationship between metal concentrations and sperm properties. ICP-MS is superior for simultaneous determination of metals and more sensitive than titration and AA. Therefore, we used ICP-MS in this study. Na and K concentrations were not determined as their ranges lay outside calibration limits, their concentrations in body fluid are well studied, and they usually maintain constant homeostatic control. The concentrations of the other 28 analyzable metals in human seminal plasma were determined. They varied in a wide range, thus, the specimens were classified into three groups (normozoospermia and oligozoospermia that were more than or under 20 × 10 6 /mL, respectively, and azoospermia) and the values compared among the three groups (Table 1). We found no significant correlation between the WHO classification in regard to sperm concentration and Zn concentration by ANOVA. Many reports have observed the importance of Se and Ca in male reproductive functions [1,[3][4][5][6][7][8], but we a: S means over 3000 µg/ml

Scattergram and regression analysis of metals in human seminal plasma
The statistical approaches favored by most reports of relationships between metals and sperm properties are the t-test [1,[3][4][5][6][7][8] and ANOVA [4,9]. However, we could not find a relationship between the three WHO groups by ANOVA. Thus, we applied the multivariate statistical method (regression analysis, PCA, and CL). Using PCA, we were able to determine the main contributing factors from many elements, including metals and sperm number. This report is the first to use the PCA approach to analyze the relationships between metals and sperm properties in human seminal plasma. The scattergrams and regression equations of all 28 metals and sperm properties (sperm concentration, motility, morphology, and semen volume) for all the samples are summarized in Figure 1. The positive correlations (r ≥ 0.250) and strong correlations (r ≥ 0.500) are represented in yellow and green, respectively. The negative correlations (r ≤ -0.250) are indicated in gray. Table 2 summarizes the metals indicative of positive and negative correlations. Metals including Ba, Co, Mo, Pb, Se, Sr, and Tl were positively and negatively correlated with at least two parameters. In particular, Se and Tl correlated positively with all the parameters examined. Zn showed a positive correlation with motility and morphology (r=0.3142 and 0.6205), whereas negative correlations with sperm concentration were weak (r = −0.2646). Thus, Zn might have positive effects on sperm properties. The heavy metals, Pb, Cd, and Cr were considered hazardous to male reproductive functions [1−5]. As expected, Pb was negatively correlated, whereas Cd and Cr showed strong correlations with concentration, motility, morphology, and volume. Statistically, trace metals including Tl and Sr showed positive correlations, although their concentrations were extremely low. Thus, their role in male reproductive function remains obscure. An increase in sample number may be necessary for detailed analyses.

Principal component analysis (PCA) of metals in human seminal plasma
PCA extracted the main contributing factors from many of the elements, including metals and sperm number. We therefore applied PCA to seminal plasma samples to analyze their relationship with the metals in detail. Thus, the sperm concentration, motility, morphology, and volume in metals were analyzed by PCA. Figure 2 details the eigen values and biplots. Most eigen values accumulated over 80% on PC10. Se was shown to highly contribute to sperm concentration, motility, morphology, and volume by biplot PC1, 2−3. Moreover, Se showed a high correlation with all sperm properties by PC2−3. By PC1−2, In and Tl showed a high correlation with volume and Zn was found to correlate with motility. The factor loadings of PCA among sperm properties and metals are shown in Table 3. From the eigen values of PC, hazardous metals in high concentration, including Cd and Cr, showed high correlations with sperm properties. The trace metals, Ce, Ga, Co, and Li showed high correlations with sperm properties. The three WHO classes were mainly determined using sperm number, followed by motility and morphology. From the biplot of metal and sperm properties, Se and Tl concentrations were found to be key metals     for the diagnosis of male infertility. From these results, we found that in addition to the well-reported metals, Se and Zn, trace metals are also important factors for evaluating sperm profiles.

Cluster analysis of metals in human seminal plasma
The results of cluster analyses among sperm number, motility, morphology, and volume and metals are shown in Figure 3. Each group was colored as follows: normozoospermia (red), oligozoospermia (blue), and azoospermia (green). Each sample was labeled by sperm number, and all samples were classified into the three main groups. However, each group was inadequately separated by sperm number as shown by the WHO categories. It was shown that sperm properties should be classified not only by sperm concentration but also by cluster analysis. These results were found to be the same using ANOVA analysis of the three WHO classes. Thus, we propose that the metal analysis approach used in this study could be used as a complement method for the diagnosis of male infertility.

Conclusion
In contrast to previous reports [1][2][3][4][5][6][7][8][9], all the metals examined in this study provided no clear correlations with the three WHO classes -normozoospermia, oligozoospermia, and azoospermia groups-as assessed by t-tests and ANOVA. However, multivariate analyses, regression analysis, PCA, and CL analysis of whole specimens revealed that metals in human seminal plasma were found to be correlated positively or negatively with various sperm properties. It was revealed that Se and Tl were key metals for the diagnosis of male infertility by PCA, and this method could be used to complement the diagnosis of male infertility. To verify the accuracy of this analytical approach for use in clinical examinations of male infertility, it is essential to accumulate a large number of specimens as well as examine as many other sperm parameters as possible.