Relationship between semen parameters and mercury concentrations in blood and in seminal fluid from subfertile males in Hong Kong

Abstract

In recent years, exposure to environmental toxins has received increasing attention as a putative cause for an apparent decline in semen quality (1Shakkebaek N.E. Giwercman A. De Kretser D. Pathogenesis and management of male infertility.Lancet. 1994; 343: 1473-1479Abstract PubMed Scopus (172) Google Scholar, 2Carlsen E. Giwercman A. Keiding N. Shakkebaek N.E. Evidence for decreasing quality of semen during past 50 years.BMJ. 1992; 305: 609-613Crossref PubMed Scopus (2212) Google Scholar). Mercury is an ubiquitous environmental toxin that has been implicated in the etiology of male infertility. Animal studies have shown that mercury induces abnormalities in sperm morphology and motility (3Lee I.P. Dixon R.L. Effects of mercury on spermatogenesis studied by velocity sedimentation cell separation and serial mating.J Pharm Exp Ther. 1975; 194: 171-181PubMed Google Scholar, 4Mohamed M.K. Burbacher T.M. Mottet N.K. Effects of methylmercury on testicular functions in Macaca fasicularis monkeys.Pharmacol Toxicol. 1987; 60: 29-36Crossref PubMed Scopus (60) Google Scholar). Results from human studies, however, are contradictory. Previous studies that failed to demonstrate a correlation between semen quality and mercury concentrations in blood and seminal plasma suffered from the limitations of a small sample size (5Chia S.E. Ong C.N. Lee S.T. Tsakok F.H.M. Blood concentrations of lead, cadmium, mercury, zinc and copper and human semen parameters.Arch Androl. 1992; 29: 177-183Crossref PubMed Scopus (74) Google Scholar), a narrow range of semen parameters (5Chia S.E. Ong C.N. Lee S.T. Tsakok F.H.M. Blood concentrations of lead, cadmium, mercury, zinc and copper and human semen parameters.Arch Androl. 1992; 29: 177-183Crossref PubMed Scopus (74) Google Scholar), and a narrow range of mercury concentrations (6Hanf V. Forstmann A. Costea J.E. Schieferstein G. Fischer I. Schweinsberg F. Mercury in urine and ejaculate in husbands of barren couples.Toxicol Lett. 1996; 88: 227-231Crossref PubMed Scopus (25) Google Scholar). An earlier pilot case-control study in Hong Kong revealed that subfertile males with sperm abnormalities had higher blood mercury concentrations than did fertile males (Choy CMY et al., unpublished observations), suggesting that our population may be at risk of mercury toxicity. The objective of our prospective observational study was to examine the relationship between semen parameters and mercury concentrations in blood and in seminal fluid of subfertile males. The Ethics Committee of the Chinese University of Hong Kong approved the study. Our study subjects were male partners of infertile couples undergoing IVF treatment at the IVF Unit of the Prince of Wales Hospital in Hong Kong between April and September 2000. Subjects with a known etiology for their semen abnormalities were excluded. Fresh semen samples obtained by masturbation, after abstinence for 3 days to 5 days, were analyzed by computer-aided semen analysis with the Hobson Sperm Tracker (HST, Hobson Tracking Systems Ltd., Sheffield, United Kingdom) as described elsewhere (7Green S. Fishel S. Hall J. Hunter A. Fleming S. Hobson G. et al.Computer image sperm selection as a novel approach to subzonal insemination in the human.Hum Reprod. 1995; 10: 1430-1434Crossref PubMed Scopus (12) Google Scholar, 8Green S. Fishel S. Morphology comparison of individually selected hyperactivated and non-hyperactivated human spermatozoa.Hum Reprod. 1999; 14: 123-130Crossref PubMed Scopus (16) Google Scholar). The frame rate was set at 50 frames per second, which analyzed the motion characteristics of sperm heads continuously for 50 points in 1 second to form a trail. After staining freshly smeared sperm slides with a modified Papanicolaou stain (Spermac, Fertility Technologies, Inc., Natick, MA), sperm morphology was determined by the HST according to the strict Kruger criteria (9Kruger T.G. Menkveld R. Stander F.S.H. Lombard C. van der Merwe J.P. van Zyl J.A. et al.Sperm morphologic features as a prognostic factor in in vitro fertilization.Fertil Steril. 1986; 46: 1118-1123Crossref PubMed Scopus (1063) Google Scholar). A technician verified all computer analyses manually. Assay of total mercury was performed on seminal fluid and whole blood by cold vapor atomic absorption spectrophotometry (Flow Injection Mercury System, Perkin Elmer Corp., Norwalk, CT). The minimum detection limit was 0.25 nmol/L and the coefficient of variation was 5.7%. Among our 111 subjects, 14 and 13 patients had mild and severe semen abnormalities, respectively. Their mean age was 38 years. None of the subjects had known occupational exposure to mercury and 32% were manual workers. Semen parameters did not vary according to job, age, or smoking habits. As shown in Table 1, seminal fluid mercury concentrations were correlated with abnormal sperm morphology, particularly head and midpiece defects, as well as with abnormal sperm motion. Straight-line velocity (VSL), linearity (LIN) of the motion path, and amplitude of lateral head displacement (ALH) were reduced, whereas average path velocity (VAP) was increased, depicting that sperm motion lost forward progression and became violently erratic in the presence of higher semen mercury concentrations. Blood mercury concentrations were significantly higher than those in semen (41.4 ± 1.7 nmol/L versus 22.1 ± 2.0 nmol/L), suggesting the presence of a functional blood-testis barrier to mercury. Neither the overall percentage of motile sperm nor sperm concentrations were correlated with mercury concentrations.TABLE 1Correlation between seminal fluid mercury concentrations and sperm morphology and motion characteristics.VariableCorrelation coefficientaSpearman’s rank correlation test.P valueAbnormal morphology0.26.02Irregular head0.49<.001Microcephaly0.24.04Abnormal midpiece0.30.01Abnormal acrosome0.03NSSperm motion characteristicsVCLbVCL (curvilinear velocity μm/s) = total distance travelled by a sperm/total tracking time elapsed.−0.008NSVSLcVSL (straight line velocity μm/s) = straight line distance travelled by a sperm/total tracking time elapsed.−0.21.03LINdLIN (linearity of the motion path) = VSL/VCL.−0.21.03ALHeALH (amplitude of lateral head displacement, μm) = mean width of sperm head oscillation.−0.20.04VAPfVAP (average path velocity μm/s) = speed along the average path of each track using an adaptive smoothing algorithm.0.21.03NS = Not significant.Choy. Semen mercury: marker of sperm toxicity. Fertil Steril 2002.a Spearman’s rank correlation test.b VCL (curvilinear velocity μm/s) = total distance travelled by a sperm/total tracking time elapsed.c VSL (straight line velocity μm/s) = straight line distance travelled by a sperm/total tracking time elapsed.d LIN (linearity of the motion path) = VSL/VCL.e ALH (amplitude of lateral head displacement, μm) = mean width of sperm head oscillation.f VAP (average path velocity μm/s) = speed along the average path of each track using an adaptive smoothing algorithm. Open table in a new tab NS = Not significant. Choy. Semen mercury: marker of sperm toxicity. Fertil Steril 2002. Our study addressed the limitations of previous studies with a larger sample size, a wider range of semen parameters, a more sensitive mercury assay, and a more reproducible and detailed method of semen analysis (10Marnet B. Vieitez G. Milhet P. Richoilley G. Lesourd F. Parinaud J. Computer assisted assessment of sperm morphology comparison with conventional techniques.Int J Androl. 1999; 23: 22-28Crossref Scopus (21) Google Scholar). Our findings are similar to those in animals exposed to mercury or other spermatotoxicant (11Rao MV. Toxic effects of methylmercury on spermatozoa in vitro.Experimentia. 1989; 45: 985-987Crossref PubMed Scopus (17) Google Scholar, 12Mohamed M.K. Lee W.I. Mottet N.K. Burbacher T.M. Laser light-scattering study of the toxic effects of methylmercury on sperm motility.J Androl. 1986; 7: 11-15PubMed Google Scholar, 13Dunson D.B. Weinberg C.R. Perreault S.D. Chapin R.E. Summarizing the motion of self-propelled cells applications to sperm motility.Biometrics. 1999; 55: 537-543Crossref PubMed Scopus (11) Google Scholar). Thus, mercury may behave as a spermatotoxicant and impair fertility potential both in vivo and in IVF programs, because fertility potential has been shown to be related to sperm morphology and motion (14Eggert-Kruse W. Schwarz H. Rohr G. Demirakca T. Tilgen W. Runnebaum B. Sperm morphology assessment using strict criteria and male fertility under in vivo conditions of conception.Hum Reprod. 1996; 11: 139-146Crossref PubMed Scopus (68) Google Scholar, 15Parinaud J. Vieitez G. Moutaffian H. Richoilley G. Labal B. Relevance of acrosome function in the evaluation of semen in vitro fertilizing ability.Fertil Steril. 1995; 63: 598-603Abstract Full Text PDF PubMed Google Scholar, 16Jeulin C. Feneux D. Serres C. Jouannet P. Guillet-Rosso F. Belaisch-Allart J. et al.Sperm factors related to failure of human in vitro fertilization.J Reprod Fertil. 1986; 76: 735-744Crossref PubMed Scopus (204) Google Scholar). This association of toxicity is biologically plausible as a causal relationship. Previous in vitro human studies have shown that sulfhydryl groups in the membrane, head, midpiece, and tail of the sperm are sites of mercury binding (12Mohamed M.K. Lee W.I. Mottet N.K. Burbacher T.M. Laser light-scattering study of the toxic effects of methylmercury on sperm motility.J Androl. 1986; 7: 11-15PubMed Google Scholar). Subsequently, sperm membrane permeability, mitochondrial functional integrity, DNA synthesis in mitotic spindles, and the microtubule sliding assembly of the sperm motor apparatus are all possible targets of mercury toxicity (3Lee I.P. Dixon R.L. Effects of mercury on spermatogenesis studied by velocity sedimentation cell separation and serial mating.J Pharm Exp Ther. 1975; 194: 171-181PubMed Google Scholar, 17Ernst E. Christensen M. Lauritsen J.G. In vitro exposure of human spermatozoa to mercuric chloride—a histochemical study.Prog Histochem Cytochem. 1991; 23: 263-268Crossref PubMed Scopus (11) Google Scholar, 18Liu C. Gao D. Preston G.M. McGann L.E. Benson C.T. Critser E.S. et al.High water permeability of human spermatozoa is mercury resistant and not mediated by CHIP28.Biol Reprod. 1995; 52: 913-919Crossref PubMed Scopus (33) Google Scholar, 19Vogel D.G. Margolis R.L. Mottet N.K. Effects of methyl mercury binding to microtubules.Toxicol Appl Pharmacol. 1985; 80: 473-486Crossref PubMed Scopus (121) Google Scholar). In addition, previous studies have shown that supporting cells such as the Sertoli and Leydig cells in the testis, as well as those outside the testis, such as in the epididymis and the seminal vesicles, are also diverse targets of mercury toxicity (20Ernst E. Moller-Madsen B. Danscher G. Ultrastructural demonstration of mercury in Sertoli and Leydig cells of the rat following methylmercuric chloride or mercuric chloride treatment.Reprod Toxicol. 1991; 5: 205-209Crossref PubMed Scopus (27) Google Scholar, 21Working P.K. Bus J.S. Hamm T.E. Reproductive effects of inhaled methyl chloride in the male Fischer 344 rat. I. Mating performance and dominant lethal assay.Toxicol Appl Pharmacol. 1985; 77: 133-143Crossref PubMed Scopus (26) Google Scholar, 22Li H. Wu Y. Yang J. Analysis of biochemical components of seminal plasma in male workers exposed to mercury vapour.Chin J Prev Med. 1995; 29: 351-353Google Scholar). We believe that an important source of mercury exposure in our population is dietary consumption of seafood, which has been shown to correlate with blood mercury concentrations in our earlier pilot study (Choy CMY et al., unpublished observations). A recent study on mercury concentration in shark fins in Thailand revealed that 6 of 10 samples had mercury concentrations exceeding the safety limit for human consumption, according to the guidelines of World Health Organization (23WildAidNew study shows shark fin consumers risk exposure to mercury [press release]. Thailand Institute of Scientific and Technological Research, Bangkok, Thailand2001Google Scholar). Other predatory fish such as tuna and swordfish may also accumulate high concentrations of mercury and are popular dietary fish species in Asia. The safety of seafood consumption in Hong Kong and in other Asian countries should be further investigated. In conclusion, our study results show that semen mercury concentrations are associated with abnormalities in sperm morphology and sperm motion in vivo. Semen, despite having a lower mercury concentration than blood, appears to be a more accurate biomarker of mercury toxicity to human sperm. The authors thank the staff of the Departments of Chemical Pathology and of Obstetrics and Gynaecology for their kind assistance in the study.