It has been hypothesized that exposure to heavy metals may impair male reproduction. To measure the effect produced by low doses of heavy metals on semen parameters, it is necessary to clarify in which body fluids those measurements must be performed. Sixty-one men attending infertility clinics participated in our study. Concentrations of lead, cadmium, and mercury were measured in whole blood, blood plasma, and seminal plasma using spectroanalytical and electrochemical methods. Semen analyses were performed according to World Health Organization criteria. For statistical analysis, Spearman's rank correlations, mean comparison tests, and discriminant analysis were calculated. Significant correlations between the measured concentrations of the three heavy metals in the same biological fluids were observed. However, no similar relationship was seen when comparing the concentrations in different body fluids of the same metal. According to our results and previous publications, seminal plasma might be the best body fluid for assessing impairment of human semen parameters.
Over time there has been a significant decline of human fertility [
These demographic transformations, as much as they are socially valued and desirable, have important clinical consequences. The fertility decline has resulted in a major delay in the average age of conception. The first pregnancy is postponed to ages at which women fecundity is decreased [
In parallel, it has been hypothesized that there is a worldwide decline in male semen quality [
Our research interests are related to the measurement of the exposure to lead (Pb), cadmium (Cd), and mercury (Hg), and its relationship with human semen quality. The main results published on that issue are summarized in Table
Review of the measurement in the exposure to lead (Pb), cadmium (Cd), and mercury (Hg) and its relation with semen quality.
Semen quality | ||||
Morphology | Motility | Sperm concentration | ||
Lead | Whole blood | Fatima 2010 [ | Telišman 2000 [ | Fatima 2010 [ |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria | (iii) 1987 criteria | (iii) 1999 criteria | ||
Mendiola 2011 [ | Fatima 2010 [ | Telišman 2000 [ | ||
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria | (iii) 1987 criteria | ||
Mendiola 2011 [ | Mendiola 2011 [ | |||
(i) | (i) | |||
(ii) | (ii) | |||
(iii) 1999 criteria* | (iii) 1999 criteria* | |||
Meeker 2008 [ | Meeker 2008 [ | |||
(i) | (i) | |||
(iii) | (ii) | |||
(iii) 1999 criteria* | (iii) 1999 criteria* | |||
Blood blasma | Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria | (iii) 1999 criteria | ||
Seminal blasma | Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria* | (iii) 1999 criteria* | ||
Hernández-Ochoa 2005 [ | Hernández-Ochoa 2005 [ | Hovatta 1998 [ | ||
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria | (iii) 1999 criteria | (iii) 1992 criteria | ||
Hernández-Ochoa 2005 [ | ||||
(i) | ||||
(ii) | ||||
(iii) 1999 criteria | ||||
Cadmium | Whole blood | Telišman 2000 [ | Mendiola 2011 [ | Mendiola 2011 [ |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1987 criteria | (iii) 1999 criteria* | (iii) 1999 criteria* | ||
Mendiola 2011 [ | Chia 1994 [ | Meeker 2008 [ | ||
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1987 criteria | (iii) 1999 criteria | ||
Chia 1994 [ | Meeker 2008 [ | |||
(i) | (i) | |||
(ii) | (ii) | |||
(iii) 1987 criteria | (iii) 1999 criteria | |||
Blood plasma | Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria* | (iii) 1999 criteria* | ||
Seminal plasma | Mendiola 2011 [ | Akinloye 2006 [ | Akinloye 2006 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria | (iii) 1999 criteria | ||
Bennof 2009 [ | Mendiola 2011 [ | Hovatta 1998 [ | ||
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1992 criteria | (iii) 1999 criteria* | (iii) 1992 criteria | ||
Bennof 2009 [ | Mendiola 2011 [ | |||
(i) | (i) | |||
(ii) | (ii) | |||
(iii) 1992 criteria | (iii) 1999 criteria* | |||
Bennof 2009 [ | ||||
(i) | ||||
(ii) | ||||
(iii) 1992 criteria | ||||
Mercury | Whole blood | Choy 2002 [ | Choy 2002 [ | Choy 2002 [ |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria | (iii) 1999 criteria | (iii) 1999 criteria | ||
Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | ||
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria* | (iii) 1999 criteria* | ||
Rignell-Hydbom 2007 [ | Rignell-Hydbom 2007 [ | |||
(i) | (i) | |||
(ii) | (ii) | |||
(iii) 1999 criteria | (iii) 1999 criteria | |||
Meeker 2008 [ | Meeker 2008 [ | |||
(i) | (i) | |||
(ii) | (ii) | |||
(iii) 1999 criteria | (iii) 1999 criteria | |||
Blood plasma | Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria* | (iii) 1999 criteria* | ||
Seminal plasma | Mendiola 2011 [ | Mendiola 2011 [ | Mendiola 2011 [ | |
(i) | (i) | (i) | ||
(ii) | (ii) | (ii) | ||
(iii) 1999 criteria* | (iii) 1999 criteria* | (iii) 1999 criteria* |
Note: This table shows author, publication year, concentration of metal in whole blood, blood plasma, and seminal plasma, their effect on semen quality parameters, and the WHO criteria used to classify the semen quality.
C: concentration of the metal, Mr: morphology, Mt: motility, SpC: sperm concentration.
*Mendiola et al. use Kruger’s strict criteria (14% of normal forms) as a cutoff for sperm morphology [
Changes in the three main semen parameters through time (1987–2010). A semen parameter was considered normal when the values were equal or above the presented figures [
1987 | 1992 | 1999 | 2010 | |
---|---|---|---|---|
Sperm concentration (×106 cells/mL) | 20–2001 | ≥20 | ≥20 | ≥15 |
Motility (%) | ≥60 | ≥50 | ≥50 | ≥40 |
Morphology (%) | ≥50 | ≥30 | ≥14 | ≥4 |
1range.
There is considerable agreement that high or even moderate concentrations of lead cause fertility problems in humans. Fatima et al. showed that >40
However, there are conflicting results about the effect on semen quality at low lead exposures. Hernandez-Ochoa and colleagues found that low lead concentrations in seminal fluid (0.2
At high concentrations, cadmium could affect semen quality. According to Akinloye et al., men with high concentrations of cadmium in seminal plasma (65
As seen with lead, there is no agreement on the effect of low concentrations of cadmium on semen quality. Telišman et al. found that even low concentrations of cadmium <1
There is clear evidence that very high concentrations of mercury in the body will harm sperm. Choy et al. showed that high concentrations of total mercury (inorganic and organic) measured in whole blood (40.6 mmol/L) resulted in <50% of progressive motility, <14% of normal morphology, and
However, Mendiola et al. did not find an alteration of motility (>50%), morphology (>14%), or sperm concentration (
There are at least two problems in assessing whether low concentrations of heavy metals have an impact on human semen quality. First of all, there are just a few studies published on that issue so far. A second problem relates to the variables measured; that is the biological samples in which the concentrations of heavy metals are measured, and the parameters used to measure semen quality (motility, morphology, and sperm concentration).
To measure the effect produced by low doses of a heavy metal in the reproductive organs, it is necessary to clarify where to perform those measurements. Concentrations of heavy metals may be measured in the whole blood, in blood plasma, and in seminal plasma. However, it is not clear whether measurements in one or another fluid are equivalent, nor to what extent there are correlations between the three measurements of these heavy metals in the different body fluids.
The objectives of this study are (1) to examine whether there are correlations between the concentrations of heavy metals (lead, cadmium, and mercury) in the three body fluids (whole blood, blood plasma, and seminal plasma) and (2) to explore whether any one of the three measures relates better than the others with the semen quality parameters.
The study population, hormone, and semen analyses have been previously described elsewhere [
A total of 181 biological samples were analyzed for Pb, Cd, and Hg, including 61 samples of seminal plasma, 61 of blood plasma, and 59 of whole blood, as two samples were lost during the study. Biological samples were dispensed into aliquots and frozen and stored at −40°C until analysis. Anodic stripping voltammetry (ASV) was used for measuring Pb and Cd concentrations. ASV was carried out using a voltamperometer with VA 663 stand and VA 608 controller (Metrohm 626, Herisau, Switzerland). The voltamperometric cell was equipped with a drop of mercury as the working electrode, an Ag/AgCl/KCl 3 M reference electrode, and a platinum auxiliary electrode.
Determination of total Hg was carried out by thermal decomposition, amalgamation, and atomic absorption spectrophotometry, using a mercury analyzer with quartz sample boats (DMA-80 Direct Mercury Analyzer, Milestone, Shelton CT, USA).
The highest grade purity reagents were employed in this procedure including nitric acid 65% and perchloric acid 70% (Suprapur, Merck, Darmstadt, Germany). The ultrapure water was purified with Millipore Simplicity 185 (Millipore GmbH, Molsheim, France) obtaining conductivity values of 0.054
In order to prepare the working standard solutions, commercially available standard solutions for Pb 1 g/L and Cd 1 g/L (Tritisol, Merck, Darmstadt, Germany) and Hg 1 g/L (Certipur, Merck, Darmstadt, Germany) were used. The limits of detection (LOD) for the body’s fluid metal levels were as follows: lead, 21
Pb and Cd determinations were performed using 0.2 mL of the biological sample deposited inside of 25 mL borosilicate glass. Acid digestion was carried out by adding 2 mL of nitric acid and 2 mL of perchloric acid and evaporating it to dryness. Once the sample was dry and cooled down, 100
Biological samples were measured by ASV according to the following method [
The statistical analysis encompassed descriptive and inferential analyses. Basic, dispersion as well as frequency parameters were calculated for descriptive analyses. Statistical analyses were performed to explore possible patterns in the concentrations of heavy metals measured in blood serum, whole blood, and seminal plasma. Spearman’s rank correlations and scatter plots were employed for comparison of variables. In the inferential analysis, the mean comparison tests and discriminant analysis were performed. All tests were two-tailed, and the level of statistical significance was set at 0.05. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).
Table
Heavy metal concentrations in seminal, blood plasma, and whole blood.
Blood plasma | ||||||
Whole blood | 9.75 (2.28) | 10.10 (7.50–11.90) | 0.10 (0.02) | 0.10 (0.09–0.12) | 1.99 (0.69) | 1.96 (1.47–2.46) |
Seminal plasma | 2.93 (0.32) | 2.90 (2.72–3.15) | 0.08 (0.01) | 0.08 (0.07–0.09) | 1.18 (0.35) | 1.13 (0.92–1.49) |
SE: standard Error, IQR: interquartile range.
Figures
(a) Relation between lead concentrations in seminal plasma and blood plasma. (b) Relation between lead concentrations in seminal plasma and whole blood. (c) Relation between lead concentrations in blood plasma and whole blood. (d) Relation between cadmium concentrations in seminal plasma and blood plasma. (e) Relation between cadmium concentrations in seminal plasma and whole blood. (f) Relation between cadmium concentrations in blood plasma and whole blood. (g) Relation between mercury concentrations in blood plasma and whole blood. (h) Relation between mercury concentrations in seminal plasma and whole blood. (i) Relation between mercury concentrations in blood plasma and whole blood.
Table
Spearman’s correlation coefficients between metal concentrations in seminal and blood plasma, and whole blood.
Blood plasma | Whole blood | ||||
Lead | Blood plasma | 0.57 | 0.67 | ||
Seminal plasma | 0.13 | 0.32 | −0.08 | 0.55 | |
Cadmium | Blood plasma | 0.14 | 0.30 | ||
Seminal plasma | 0.12 | 0.36 | −0.50 | 0.72 | |
Mercury | Blood plasma | 0.17 | 0.19 | ||
Seminal plasma | −0.13 | 0.34 | −0.34 | 0.80 |
Figures
(a) Relation between lead and cadmium concentrations in whole blood. (b) Relation between lead and mercury concentrations in whole blood. (c) Relation between cadmium and mercury concentrations in whole blood. (d) Relation between lead and cadmium concentrations in blood plasma. (e) Relation between lead and mercury concentrations in blood plasma. (f) Relation between cadmium and mercury concentrations in blood plasma. (g) Relation between lead and cadmium concentrations in seminal plasma. (h) Relation between lead and mercury concentrations in seminal plasma. (i) Relation between cadmium and mercury concentrations seminal plasma.
Spearman’s correlation coefficients and scatter plots revealed a high correlation between the concentrations of the three metals in the same biological fluids. Table
Spermean’s correlation coefficients between seminal plasma, blood plasma, and whole blood, with metal concentrations.
Cadmium | Mercury | ||||
Seminal plasma | Lead | 0.740 | 0.001 | 0.760 | 0.001 |
Cadmium | 0.870 | 0.001 | |||
Blood plasma | Lead | 0.550 | 0.001 | 0.750 | 0.001 |
Cadmium | 0.700 | 0.001 | |||
Whole blood | Lead | 0.850 | 0.001 | 0.950 | 0.001 |
Cadmium | 0.792 | 0.001 |
To explore whether these correlations were determined by associations with other factors, exploratory scatter plots were generated between the concentrations of the three metals in the three biological fluids and possible confounding variables. Possible confounders were such as “occupation,” “tobacco smoke,” “exposure to toxics at work” or “using metals at work.” No patterns were observed. Hypothesis tests were used to detect significant differences in the mean concentrations of metals and the possible confounding factors used in the scatter plots. Not significant differences were found (data not shown).
As a final alternative, metal concentrations were categorized in two, three, and four groups using the mean values, tertiles, and quartiles, respectively. Discriminant analysis was then used to detect whether any of the factors was related to the categories of the metal concentrations. To this end, different discriminant analysis evaluating the overall Wilks’ lambda and the owners of each factor were produced, but none of them were satisfactory.
Using the Spearman’s correlation coefficient and scatter plots revealed a high correlation between the measured concentrations of the 3 heavy metals in the same biological fluids. However, no similar relationship was observed when comparing the concentrations in different body fluids of the same metal.
It would be reasonable to expect that subjects with high and low levels of exposure to any metal would show similar positions (low or high concentrations) in the measurements made in any body fluid. However, we found no correlation between the concentrations of any of the metal in the three biological samples analyzed (whole blood, blood plasma, and seminal plasma).
Other authors, similarly, found no correlation between the concentrations of the same metal in different fluids [
There are some possible hypotheses for these phenomena. The three heavy metals are bound and transported by erythrocytes [
Surprisingly, the concentrations of Pb, Cd, and Hg were correlated in the same biological samples. Howatta et al. also found that the concentrations of cadmium and lead in seminal plasma were correlated [
Correlations of the three heavy metals in the same body biological fluid may be due to an interaction between the different metals in the same compartment, so that the concentration of one metal determines the concentration of the others. We are not aware of lead, cadmium, or mercury modulate each other. However, it has been published that selenium produces the redistribution of Hg from plasma to erythrocytes at higher ratio [
As to how to measure the effect produced by heavy metal concentrations on semen quality, it would be better to measure those metals in seminal plasma than in blood plasma or whole blood. Heavy metal concentrations in blood samples do not necessarily reflect the seminal plasma ones, since heavy metal concentrations reaching the seminal plasma could be quite different.
Heavy metals have a strong capacity to induce oxidative stress in body cells by disintegration of the lipid membrane, and spermatozoa are quite sensible to oxidative stress [
Furthermore, as it can be seen in Table
Finally, our findings might be attributed to chance or bias. The sample of individuals included in the study was small and the lack of statistically significant correlations may be a consequence of that. Our findings are, however, consisting with those [
Our study suggests that there is no correlation between the concentrations of any of the metals in the three biological samples analyzed (whole blood, blood plasma, and seminal plasma) and there is a correlation between the concentrations of Pb, Cd, and Hg in the same biological samples. According to our results and previous publications, seminal plasma might be the best body fluid for assessing impairment of human semen parameters.
The authors declare that they have no competing interests.
The authors are grateful for the assistance of Mr. Lorenzo Vergara Pagán for specimen handling and heavy metal analysis. This research project was partially supported by Fundación Séneca, Agencia Regional de Ciencia y Tecnología, Region de Murcia (Ref: 00694/PI/04), the Reproductive Medicine Chair of the Miguel Hernández University-Instituto Bernabeu, Fondo de Investigación Sanitaria, and Gestión Clínica Avanzada.