Associations of semen quality with non-essential heavy metals in blood and seminal fluid: data from the Environment and Male Infertility (EMI) study in Lebanon

Study design

Participants were categorized into two groups, low- and normal-quality semen groups, according to the World Health Organization (WHO) 4th edition reference value of semen quality [32]. Participants with a semen volume < 1.5 mL, sperm concentration < 15 million/mL, total count < 39 million, progressive motility < 32%, viability < 58%, and/or normal WHO morphology < 30% were assigned to the low-quality semen group A. Participants whose semen analyses expressed better results in all the above parameters were assigned to the normal-quality semen group B. A questionnaire was completed by each participant. Questions included information on socio-demographic characteristics, personal lifestyle (tobacco, alcohol, and diet), occupational exposure, medication intake, and medical history.

Specimen collection

Whole venous blood samples were collected using stainless steel needles into tubes containing ethylene diamine tetra-acetic acid. Blood and semen specimens were stored at − 70 °C for subsequent metal analysis.

Following an abstinence period of 3–7 days, each participant provided a semen sample into a sterile wide-mouth metal-free polypropylene container at the Fertility Center by masturbation. Participants were asked to wear metal-free gloves while providing semen samples. All metal components in the semen collection room were carefully covered with plastic material. Semen samples were handled and analyzed by two qualified laboratory technologists according to the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Mucous Interaction [33]. The semen volume (mL) was measured using a metal-free polypropylene graduated pipette. Sperm concentration (million/mL) and progressive motility (%) were determined manually using a Makler® counting chamber (Irvine Scientific, CA). Total sperm count (million) was calculated as sperm concentration × semen volume. Sperm morphology was determined by high-power magnification (× 1000) on air-dried smears stained with a Wright-Giemsa stain based on the WHO guidelines (1999) guidelines [33]. Laboratory technicians analyzed two slides for each participant with no less than 100 sperms per slide. External quality controls were used, and discrepancies in results were reported to be insignificant. The overall laboratory CV for inter-observer determinations of sperm morphology was ~ 17%.

Metal analyses

Whole blood samples (2 mL) were digested with 70% nitric acid (HNO₃) and oxygen peroxide (H₂O₂), filtered and diluted to 10 mL. Seminal fluid aliquots (0.5 mL) were digested with 70% nitric acid (HNO₃) (Fisher Scientific, USA), filtered, and diluted to 5 mL. Digestion was performed in a closed vessel microwave system (temperature program reaching 120 °C and sustained for 10 min at 1000 W). Double distilled purified deionized water from Milli-Q system was used for dilution before performing the analyses. All glassware was washed and immersed in concentrated HNO₃ overnight and then rinsed with deionized water before use.

Metal levels (Pb, Cd, As, Hg, Ba, and U) in blood and seminal fluid were analyzed using an ion-coupled plasma-mass spectrometry (ICP-MS; Agilent 7500ce, Agilent Technologies, Germany) equipped with a cell dynamic range (CDR). For quality control, certified reference materials were used as per manufacturer, and measurements of analytes found to be within standard range. Quantification of analytes was done by the standard addition of 0, 1, 5, and 10 ppb of heavy metals. Responses were measured for the series of the standards added, and results were plotted then extrapolated to y = 0 to get the value of the metal. To assess instrument performance, the standard reference material was analyzed after each 20 samples. One blank tube containing deionized water was added to each batch of samples. Metal levels in all blank samples were lower than the limits of detection (LOD). The LOD for Hg was 0.5 and 1 μg/L for all other metals. For statistical purposes, no censoring of concentrations below the detection limit was implemented [34]. Machine read values were analyzed as described and were reported in micrograms per liter.

Blood and seminal fluid metals

Analyte concentrations stratified by semen quality reference values were compared in the blood and seminal fluid of participants (Table ​(Table2).2). Analytes measured in blood did not significantly differ between groups on the basis of semen quality. Conversely, we found that participants with low-quality semen had significantly higher Cd and Ba concentrations in the seminal fluid than participants with normal-quality semen. For the remaining metals (Pb, As, Hg, and U), no significant differences were found in the seminal fluid of either groups. Spearman’s rank correlation analyses showed positive correlations between blood and seminal fluid concentrations of analytes measured, with the strongest correlation coefficients observed for Cd (r = 0.67) and Ba (r = 0.58).

When comparing analyte concentrations in the blood and seminal fluid within same participants (Table ​(Table3),3), we found Pb to be significantly more concentrated in blood than in the seminal fluid. Conversely, both As and U were significantly more concentrated in the seminal fluid than in the blood of participants. We detected no significant differences between both compartments for the remaining elements (Cd, Ba, and Hg).

Lead

In the present study, we found significant associations of seminal Pb concentrations with below-reference sperm viability. Such relationship could not be demonstrated for blood Pb, however, despite the detection of significantly higher concentrations of lead in blood compared to seminal fluid. Current evidence indicates that associations between non-occupational lead exposure and semen quality remain inconclusive in men. Consistent with our findings, Mendiola et al. observed significant positive associations between seminal Pb and low sperm motility in a Spanish clinic-based population of infertile men [28]. Doubling in seminal Pb was also found to be associated with a 47% lower total motile sperm count [37]. These findings were not reproducible for Pb levels in blood. In a Mexican low-level exposure community-based study, Hernández-Ochoa et al. also supported an impairment of sperm progressive motility and morphology with elevated seminal Pb concentrations but failed to associate Pb in blood with semen quality measures [16].

It should be noted nonetheless that the use of seminal Pb measurements as a biological marker of toxicant exposure in men is not universal. Data on this subject are controversial particularly when considering the nature of the population exposure. In their study of lead smelter workers in British Columbia, Alexander et al. concluded that lead concentrations in the blood of occupationally exposed men were more consistently associated with indicators of spermatogenesis than in seminal plasma [38]. Similarly, Robins et al. reported no associations between semen lead and sperm quality in South African Lead Acid Battery Plant workers [39]. Telisman et al. also concluded that blood indicators of lead exposure are superior to seminal fluid indicators in industrial workers with regard to predicting decreased semen quality [19]. Taken together, these findings suggest that non-occupational low-level lead exposure in the reproductive system may not be best reflected by blood measurements and that Pb would be more appropriately assessed in the seminal fluid in this population of men. Additional research is nonetheless required to confirm this contention.

Animal experimental studies demonstrated dose-dependent reductions in testicular alkaline phosphatase and sodium-potassium ATPase activities in testicular tissues of rats exposed to lead [40]. Production of reactive oxygen radicals has also been detected leading to increased lipid peroxidation, loss of plasma membrane fluidity, and decreased sperm motility [41].

In an attempt to reconcile discrepancies with reported studies, we compared the concentrations of lead in our study with published data from populations with environment low-level exposures. In the current study, the observed mean blood Pb concentrations in Lebanese men attending an infertility clinic were 44.43 ± 42.96 μg/L. These values were lower than those reported by Mendiola et al. (101 μg/L) on a similar clinic-based population of Spanish men [28] and by Hernandez-Ochoa et al. (93 μg/L) on a community-based population of men from Mexico [16]. Variations between studies could be accounted for not only by variable exposure levels but also by differences in methodologies and laboratory measurements techniques. Taken together, the relatively lower lead blood levels found in Lebanese men indicate that lead exposure in the country ranks low on the priority scale of public health perspectives.

Cadmium

In the present study, we found Cd in blood and seminal fluid to be significantly associated with low sperm viability. While the relationship between non-occupational Cd exposure and semen function remains controversial, numerous observational studies found significant negative associations of seminal Cd and sperm motility/viability [15, 28, 42, 43]. Fewer studies reported similar correlations between blood Cd and low sperm concentration [44]. In disagreement with the preceding, other investigators however failed to validate any significant relationships between blood and/or seminal Cd and any sperm quality parameters [37, 45–47].

Experimental animal models, on the other hand, demonstrated that Cd exposure is toxic to spermatogenesis leading to decreased sperm motility and concentration [48–50]. Cadmium, by competing with calcium for calmodulin binding, is believed to interfere with protein tyrosine phosphorylation [41]. Also, by increasing peroxidation of membrane lipids, Cd was shown to reduce the phosphorylation of axonemal proteins. Experimental human studies laid further evidence in favor of an adverse effect of Cd on sperm function. When a cohort of 210 fertile and infertile men was exposed to cadmium, significant drops in sperm motility and viability were observed in a time- and dose-dependent fashion [51]. A detrimental effect of Cd on sperm metabolism is found and is believed to be mediated by inhibition of glycogen phosphorylase, glucose-6-phosphatase, Mg-dependent ATPase, and succinic acid dehydrogenase [52].

Blood Cd concentrations were determined to be 0.4–4 μg/L in occupationally non-exposed men [53] and to exceed 10 μg/L in environmentally contaminated areas [53]. The mean blood Cd in the present study was 20.96 ± 45.81 μg/L, which is clearly indicative of a significant environmental exposure. Sources of contamination in Lebanon may be traced back not only to catastrophic cycles of demolitions as a result of repeated wars over the past decades but also to the ongoing construction boom in the country with an exponential growth of unregulated quarries amidst populated areas with the aim of increasing demands for construction aggregates. Taking into account the prolonged estimated biological half-life of Cd of 10–40 years in humans and its slow 0.01% daily body burden excretion [6], these data underline an important public health issue and constitute an important database platform on which future governmental regulations could be based.

Barium

In the current study, Ba levels in blood and seminal fluid of participants correlated negatively with low sperm viability. There is, to date, no reliable information about reproductive health hazards in men or experimental animals exposed to barium. Disruptions to spermatogenesis have been observed in rats exposed to inhaled Ba carbonate [54]. Disturbances appeared reversible in nature and involved decreased sperm count, motility, and osmotic resistance. In contrast, rats and mice subjected to intermediate and chronic oral barium chloride exposure had no associated histopathological changes in their testes and no alterations in epididymal sperm counts, motility, or morphology [55].

Barium is an alkaline metal ubiquitously present in nature, including volcanic earth crust and drinking water. Soluble barium compounds (BaCl₂ and BaCO₃) are known for their acute general health toxicity. Epidemiological data examining the effects of chronic low-level Ba exposure on spermatogenesis are sparse despite the increased use of this compound in industry and medicine. The current study represents one of very few human studies examining the effects of chronic low dose Ba exposure on reproduction in men. More research nonetheless is needed to develop a more explicit understanding of barium accumulation in male reproductive organs and its potential reproductive implications.

Uranium

The findings of this study indicate notable uranium exposure of the participants, with significantly more measurable U concentrations in the seminal fluid than in blood. Also, significant associations of seminal U levels were observed with below-reference progressive sperm motility, viability, and normal morphology. In contrast, blood U levels did not show similar relationships with any sperm quality parameters. While toxicity as a result of natural uranium is extremely rare, exposure to depleted uranium, a man-made by-product of the uranium enrichment process, has become a significant public health concern lately, mainly in war-inflicted zones [56]. While the U by-product is partially depleted of its radioactivity, it retains its chemical toxicity as a non-essential heavy metal. The findings of the present study are exceptional, in that very few other studies investigating the effects of environmental uranium exposure on the reproductive system of men are available. In their research of veterans wounded at the First Gulf War, Squibb and McDiarmid failed to demonstrate any effects of depleted uranium exposure on sperm concentration and motility in men [56]. Whereas experimental studies evaluating the effects of natural U on the male reproductive system in rodents have demonstrated a clear association with testicular atrophy and reduced spermatogenesis [57], animal data derived from depleted U exposure are far more controversial. In one study, depleted U in rodents was found to pass through the blood-testis barrier by modulating the gene expression of molecules involved in the regulation of tight junctions and to accumulate in reproductive organs, seminal fluid and spermatozoa [58]. In the same study and in others [58–60], the presence of depleted U in the testes of adult rats did not induce oxidative stress [58] and did not correlate with any defects in spermatogenesis namely sperm concentration and velocity [59, 60]. An endocrine-mediated effect was nonetheless observed in rats as testicular testosterone production decreased along with depleted U exposure [58]. In contrast, other investigators demonstrated a dose-dependent increase in sperm abnormalities in rodents exposed to depleted U, with evidence of histologic atrophy of convoluted tubules, reduction in spermatogenic cells, and increase in sperm DNA damage [61].

In Lebanon, possible sources of human exposure are derived from the use of depleted uranium munitions during repeated rounds of war violence. Once fired, these munitions produce small dust particles and large shrapnel fragments, contaminating air and soil. While air inhalation is considered the main route of human exposure, more recent reports have favored soil as a vehicle of contamination of the food chain and underground waters [62]. This study suggests that the use of blood U measurement as indicator of chronic body burden may underestimate the duration and nature of exposure. Since depleted U has been shown to accumulate in body organs, namely in kidneys and testes, seminal fluid levels seem to be more appropriate for the estimation of cumulative chronic exposures [62]. From a public health point of view, it is unclear how low-dose chronic exposure should be managed at the population level bearing in mind (a) the absence of a clear definition of what would constitute such exposure in humans and (b) the non-existence of a comprehensive approach for large-scale detoxification. As more humans are being exposed to depleted U, there is a fundamental need for large epidemiologic studies with the aim to examine the nature of the threat to general and reproductive health.