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Cryptorchidism, hypospadias, subfertility, and testicular germ-cell tumor have been suggested to comprise a testicular dysgenesis syndrome (TDS) based on the premise that each may derive from perturbations of embryonal programming and gonadal development during fetal life. Endocrine-disrupting chemicals have been hypothesized to be associated with these disorders given the importance of sex steroid hormones in urogenital development and homeostasis. Organochlorines are one such set of compounds which are defined as containing between one and ten covalently bonded chlorine atoms. These compounds are persistent pollutants with long half-lives, accumulate in adipose tissue when ingested, bioaccumulate and biomagnify, and have complex and variable toxicological profiles. Examples of organochlorines include dichlorodiphenyltrichloroethane (DDT) and its metabolites, polychlorinated biphenyls (PCBs) and chlordane. In this comprehensive review of human epidemiologic studies which have tested for associations between organochlorines and facets of TDS, we find evidence for associations between the exposures p,p′-DDE, cis-nonachlor, and trans-nonachlor with TGCT. The sum of the evidence from human epidemiologic studies does not indicate any association between specific organochlorines studied and cryptorchidism, hypospadias, or fertility. Many other endocrine-disrupting chemicals, including additional organochlorines, have yet to be assessed in relation to disorders associated with TDS, yet study of such chemicals has strong scientific merit given the relevance of such hypotheses to urogenital development.
In 2001, Skakkebaek et al. proposed that various disorders of male reproductive health, namely cryptorchidism, hypospadias, subfertility, and testicular germ-cell tumor (TGCT), derived from perturbations of embryonal programming and gonadal development during fetal life and thus comprised a testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001). Although it remains contentious whether these disorders constitute a syndrome (Akre & Richiardi, 2009; James, 2010; Jorgensen et al., 2010; Wohlfahrt-Veje et al., 2009), it is conceivable that endocrine-disrupting chemicals could play an etiological role given the importance of sex steroid hormones in urogenital development and homeostasis (Toppari, 2008).
Organochlorines are one such set of endocrine-disrupting chemicals which have been hypothesized to be associated with facets of TDS. Organochlorines contain between one and ten covalently bonded chlorine atoms, which confer very long half-lives (Bennett et al., 1974; Seegal et al., 2010; Wolff et al., 2000). Organochlorines are also highly insoluble in water and accumulate in adipose tissue when ingested. Combined, these properties make organochlorines persistent pollutants that bioaccumulate and biomagnify. The determinants of their toxicity are complex and, although many organochlorines are approved for use, others are known to affect homeostatic processes or are highly reactant (Kaushik & Kaushik, 2007).
The organochlorine compounds most extensively studied have been dichlorodiphenyltrichloroethane (DDT) and its metabolites, and the polychlorinated biphenyls (PCBs). DDT was first synthesized in 1874 and was subsequently discovered to have insecticidal qualities in 1939. General commercial use of DDT as an anti-malarial and agricultural pesticide became widespread from 1945 onwards. Subsequent research showing toxicity and long-term persistence led to DDT being banned in many countries, beginning in the early 1970s. Agricultural use of DDT is now banned by the Stockholm Convention on Persistent Organic Pollutants (United Nations Environment Programme (UNEP), 2001), although DDT continues to be used as an anti-malarial in parts of Africa and Asia. Constituents of commercial grade DDT are the isomers p,p′-DDT (~77%) and o,p′-DDT (~15%), as well as dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) (United Nations Environment Programme (UNEP), 1979). DDT is known to be estrogenic (White et al., 1994), whereas its primary persistent metabolite, DDE, has been shown to have anti-androgenic properties (Kelce et al., 1995). The International Agency for Research on Cancer (IARC) classifies DDT as a Group 2B carcinogen (possibly carcinogenic to humans) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1992).
PCBs are a group of related compounds (congeners) composed of two carbon-linked benzene rings to which are attached between 1 and 10 chlorine atoms. Used as dielectric fluid, flame retardants, ink solvents, and plasticizers, PCBs were first manufactured commercially in the late 1920s. Evidence of their toxicity led to the institution of bans in many countries, beginning in the late 1970s and to their inclusion on the list of compounds in the Stockholm Convention on Persistent Organic Pollutants treaty of 2001 (United Nations Environment Programme (UNEP), 2001). PCBs are known to be able to cause hormonal perturbations and have been associated with urogenital maldevelopment in animal models (Toppari, 2008; Toppari et al., 1996). Several schemata have been suggested in order to organize PCB congeners into groupings based on biological activity and chlorine substitution patterns (Wolff et al., 1997; Wolff & Toniolo, 1995); congener groups with more pronounced sex steroid effects may be most relevant for disorders which comprise TDS (Toppari, 2008). The IARC classifies PCBs as Group 2A carcinogens (probably carcinogenic to humans) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1987).
Other organochlorines such as cyclodienes (chlordane, heptachlor, dieldrin, mirex), polychlorinated dibenzofurans (PCDF), toxaphene, hexachlorobenzene (HCB), and hexachlorocyclohexane (HCH) have also been assessed with disorders encompassed by TDS. The IARC classifies each of these compounds as Group 2B carcinogens (possibly carcinogenic to humans) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 1987; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2001).
In this review, we will summarize the human epidemiologic evidence with regard to the relationships between organochlorines and facets of TDS. Studies for inclusion were identified using the following terms in a search of the PubMed database on 29th October 2010: organochlorine, EDC, endocrine-disrupter, endocrine-disrupting, dichlorodiphenyltrichloroethane, DDT, dichlorodiphenyldichloroethylene, DDE, polychlorinated biphenyl, PCB, testicular dysgenesis syndrome, spermatogenesis, semen quality, semen parameters, sperm quality, infertility, hypospadias, cryptorchidism, testicular germ-cell tumors, and testicular cancer.
Two congenital anomalies are included in the definition of the TDS: cryptorchidism and hypospadias. Cryptorchidism is the failure of one or both testicles to descend into the scrotum. Testicular descent occurs during two phases of in-utero life. The first phase occurs during first trimester when the testes migrate from the intra-abdominal space to the top of the inguinal ring. The testes remain at the top of the inguinal ring until late in third trimester when they migrate through the inguinal ring into the scrotal sac (Hutson et al., 2010). In some cases, this final migration does not occur until after birth so cryptorchidism cannot be ascertained reliably at the time of birth. Evidence indicates that the vast majority of testes that will descend spontaneously will do so by 6 months of age (Hutson et al., 2010), thus study designs that rely only on diagnosis in the delivery room are sub-optimal. Hypospadias, the condition in which the opening of the urethra is on the ventral side of the penis rather than that at the tip of the glans penis, can be diagnosed reliably at birth. Hypospadias arise during the first trimester of in-utero life and are classified as mild (1st degree) to severe (3rd degree), depending on where the urethra opens on the penis.
Eight studies have examined the relationship between cryptorchidism and/or hypospadias and DDT and/or metabolites of DDT (Table 1). Five of the studies used maternal sera (Bhatia et al., 2005; Carmichael et al., 2010; Giordano et al., 2010; Hosie et al., 2000; Longnecker et al., 2002), while one used breast milk (Damgaard et al., 2006), one used placenta (Fernandez et al., 2007) and one examined both cord blood and colostrum (Brucker-Davis et al., 2008). The largest of the studies was nested in the prospective Collaborative Perinatal Project (CPP). The CPP enrolled approximately 42,000 pregnant women at 12 medical centers in the U.S. between 1959 and 1966 (Niswander et al., 1972). Among the 22,347 boys born during the study, there were 241 cases of cryptorchidism and 214 cases of hypospadias. Longnecker and colleagues (Longnecker et al., 2002) contrasted serum p,p′-DDE levels among mothers of the cases with levels among 552 mothers of boys without either condition. No association between p,p′-DDE and either anomaly was evident (cryptorchidism: Q5:Q1 odds ratio (OR)=1.3, 95%CI=0.7–2.4; hypospadias: Q5:Q1 OR=1.2, 95%CI=0.6–2.4). Using a similar prospective study design, Bhatia and colleagues examined the association between maternal serum levels of p,p′-DDT and p,p′-DDE and cryptorchidism and hypospadias in the prospective Child Health and Development Studies (CHDS) (Bhatia et al., 2005). Conducted in Northern California, the CHDS enrolled 20,754 pregnancies between 1959 and 1967. The investigators examined serum levels among the mothers of 75 boys with cryptorchidism, 66 boys with hypospadias and 283 boys with neither condition. There was no association between p,p′-DDT or p,p′-DDE with either outcome.
Smaller studies of DDT and the congenital anomalies have been reported from Germany (Hosie et al., 2000), Denmark and Finland (Damgaard et al., 2006), Spain (Fernandez et al., 2007), France (Brucker-Davis et al., 2008), Italy (Giordano et al., 2010) and the U.S. (Carmichael et al., 2010). Hosie et al (Hosie et al., 2000) examined maternal serum levels of p,p′-DDT, o,p′-DDT and p,p′-DDE in 18 mothers of cryptorchid boys and 30 mothers of controls and reported no significant associations. Damgaard et al. (Damgaard et al., 2006) examined the same three compounds in the breast milk of mothers of cryptorchid (n=62) and non-cryptorchid boys (n=68) in Denmark and Finland and, similarly, found no associations. Brucker-Davis et al (Brucker-Davis et al., 2008) and Giordano et al (Giordano et al., 2010) each examined p,p′-DDE and reported no significant association with either cryptorchidism (Brucker-Davis et al., 2008) or hypospadias (Giordano et al., 2010). Fernandez et al (Fernandez et al., 2007) examined cryptorchidism and hypospadias jointly and reported no association either p,p′-DDT or o,p′-DDT. Carmichael et al (Carmichael et al., 2010), using maternal sera, examined the association with hypospadias and reported no association with either p,p′-DDT or p,p′-DDE.
Five of the eight studies that examined DDT and its metabolites also examined PCBs (Table 2) (Brucker-Davis et al., 2008; Carmichael et al., 2010; Giordano et al., 2010; Hosie et al., 2000; McGlynn et al., 2009). One additional study, conducted in the Faroe Islands of Denmark, also examined the relationship between PCBs and cryptorchidism (Mol et al., 2002). The largest of the PCB studies (n=230 cryptorchidism, n=201 hypospadias, n=593 controls) was, again, the nested case-control study conducted within the CPP (McGlynn et al., 2009). Examining individual PCB congeners and sum of PCBs in maternal sera, the investigators found no significant associations. Hosie et al. (Hosie et al., 2000), Giordano et al (Giordano et al., 2010) and Carmichael et al.(Carmichael et al., 2010) also examined PCBs in maternal serum and none of the three studies reported significant differences in either cryptorchidism (Hosie et al., 2000) or hypospadias (Carmichael et al., 2010; Giordano et al., 2010). Mol et al (Mol et al., 2002) examined PCBs in umbilical cord tissues, while Brucker-Davis et al (Brucker-Davis et al., 2008) examined PCBs cord blood and colostrum. Neither study reported an association between PCBs and cryptorchidism. In sum, none of the six reported studies found an association between PCBs and either cryptorchidism or hypospadias. While most of the studies were rather small and some were conducted in more recent years when PCB levels were lower, the CPP study (McGlynn et al., 2009) included over two hundred cases each of cryptorchidism and hypospadias and almost 600 controls and examined sera from the 1960s when PCB levels were high. As with the other studies, the CPP found no associations.
In addition to examining DDT, DDE and PCBs, several of the previously mentioned studies have examined other organochlorine compounds in relationship to cryptorchidism and, to a lesser extent, hypospadias. Studying cryptorchidism, Hosie and colleagues (2000) examined a total 26 compounds while Damgaard and colleagues examined 21 compounds. Hosie et al. found significant associations with two of the studied compounds: heptachlor epoxide and hexachlorobenzene. Neither Damgaard and colleagues (2006), nor Pierik and colleagues (2007) however, found associations with heptachlor epoxide or hexachlorobenzene. In contrast, Damgaard et al. (2000) did find a significant association with trans-chlordane, a finding not replicated by Hosie et al. (2000). Fernandez et al. (2007), studying a total of 18 compounds, found a significant association with mirex and hexachlorocyclohexane. The hexachlorocyclohexame association was not replicated in the studies of Pierik et al. (2007), Damgaard et al. (2000), or Hosie et al. (2000). Studying hypospadias, Giordano et al reported a significant association with hexachlorobenzene, a finding which was not replicated by Carmichael et al. (2010).
In summary, none reported a significant association with p,p′-DDT, o,p′-DDT or p,p′-DDE and either cryptorchidism or hypospadias. As the levels of DDT and its metabolites have been declining among populations of developed countries in recent years (Rigét et al., 2010), it is unlikely that future studies will find a relationship not seen in the older studies. Similarly, at the present time, the accumulated data do not support an association between PCB exposure and either cryptorchidism or hypospadias. As with DDT levels, levels of PCBs in developed countries have been declining in recent decades (Rigét et al., 2010), thus future studies of newborns will likely report lower exposures than those seen in the current literature. While some studies have reported associations with other organochlorine compounds, thus far no positive association has been replicated. The small number of cases and controls and the large number of compounds examined in a majority of studies argue that any findings should be interpreted with caution.
Several reviews have reported international trends in semen quality in the last half of the twentieth century. Declines in sperm concentration in the U.S., Europe, and Australia were reported in a systematic review of 101 studies between 1934 and 1996 (Swan et al., 2000). This study reinforced the conclusion of an earlier systematic review of 61 studies conducted between 1938 and 1991 (54 of which were included in Swan et al.) that support the existence of significant declines in sperm concentration and decreases in mean seminal volume in men with no history of infertility (Carlsen et al., 1992).
Spermatogenesis, including testicular development and maturation, may be affected by endocrine-disrupting chemicals during several critical stages of development, specifically in-utero development and puberty. The largest number of studies of organochlorines and spermatogenesis have been conducted on adult populations and can be classified into two categories: (1) studies that have primarily consisted of general population or presumed fertile population samples (Aneck-Hahn et al., 2007; De Jager et al., 2006 p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study; Dhooge et al., 2006; Jonsson et al., 2005; Richthoff et al., 2003; Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function; Toft et al., 2006); and (2) studies conducted in infertility or andrology clinic settings that evaluated associations between organochlorines and spermatogenesis comparing groups of men with male factor infertility with men from couples identified with female factor infertility, which on occasion also include a subgroup of men with idiopathic infertility (Charlier & Foidart, 2005; Dallinga et al., 2002; Hauser et al., 2003 p’-DDE; Magnusdottir et al., 2005; Pant et al., 2004; Rozati et al., 2002). Most of these existing studies were largely cross-sectional in design with serum or plasma collected at the same time as semen.
To date, nine studies have examined the association between DDT and/or DDT metabolites and spermatogenesis (Table 3), including four studies of presumed fertile populations and five infertility/andrology clinic-based studies (De Jager et al., 2006 p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study; Dhooge et al., 2006; Jonsson et al., 2005; Richthoff et al., 2003; Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function; Toft et al., 2006). In a cross-sectional evaluation of East and West coast Swedish fishermen (n=195), Rignell-Hydbom and colleagues examined the association between serum levels of p,p′-DDE and testis volume, semen volume, sperm count, concentration, and motility (Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function). After adjusting for age, serum p,p′-DDE was not associated with any of the measured semen parameters (Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function).
In a geographic comparison of 798 fertile men in four regions in Europe (Warsaw, Poland – low PCB/moderate DDE; Kharkiv, Ukraine – low PCB/high DDE; Greenland – high PCB/high DDE; Sweden – high PCB/low DDE), Toft and colleagues evaluated serum p,p′-DDE concentration and regional differences in semen quality (Toft et al., 2006). The geometric means of percent motile sperm were significantly lower in Greenland (55%; 95% confidence interval (CI): 52%–58%) and the Ukraine (54%; 95% CI: 52%–57%) compared with Poland (60%; 95% CI: 57%–63%), suggesting that higher levels of DDE exposure may be associated with impaired sperm motility. The study provided little evidence that DDE exposure was related to sperm count, concentration or morphology (Toft et al., 2006).
In a North American study, the association of seminal parameters (semen volume, sperm count, concentration, motility and morphology) and plasma p,p′-DDE level was evaluated among 116 young men residing in Chiapas, Mexico, a location with endemic malaria that has a history of household DDT exposure (De Jager et al., 2006 p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study). Plasma p,p′-DDE was positively associated with increased tail defects (beta = 0.003; p-value = 0.017) and negatively correlated with sperm motility (beta = −8.38; p-value = 0.05) (Ayotte et al., 2001; De Jager et al., 2006 p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study). No associations were found between serum p,p′-DDE and semen volume, sperm count or sperm concentration (De Jager et al., 2006 p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study).
In a South African study, the association of semen quality (semen volume, sperm count, concentration, motility and morphology) and serum p,p′-DDE and p,p′-DDT levels evaluated among 303 healthy males between 18 and 40 years of age recruited from 3 communities in Limpopo Province, endemic malaria areas sprayed with DDT annually (Aneck-Hahn et al., 2007). Utilizing age-adjusted linear regression, lipid-adjusted serum p,p′-DDE was inversely associated with semen volume (beta = −0.0003; 95% CI: −0.0006, −0.00004) and total sperm count (beta = −0.003; 95% CI: −0.006, −0.0002) (Aneck-Hahn et al., 2007). Serum p,p′-DDE levels were not associated with sperm concentration, percent progressive motility or percent motile sperm, and percent normal morphology. No associations were found between lipid-adjusted serum p,p′-DDT and semen volume, sperm count, sperm concentration, percent progressive motility or percent motile sperm, and percent normal morphology (Aneck-Hahn et al., 2007). Measuring sperm motility with the Hamilton Thorne Computer Assisted Sperm Analysis (CASA) system, mean CASA motility were inversely associated with serum p,p′-DDT and p,p′-DDE concentrations; however, other measures of sperm motility demonstrated no association (Aneck-Hahn et al., 2007).
Of the five studies recruiting subjects from infertility or andrology clinics, those based in The Netherlands, Iceland or Belgium, report no association between serum measures of DDT and/or p,p′-DDE and semen characteristics (Charlier & Foidart, 2005; Dallinga et al., 2002; Magnusdottir et al., 2005). Specifically, in The Netherlands, Dallinga and colleagues compared 34 men with abnormal sperm quality with 31 men with “normal” sperm quality and proven fertility (Dallinga et al., 2002). The authors investigated associations of serum concentrations of p,p′-DDT and p,p′-DDE with semen volume, sperm count, concentration, motility, and morphology and reported no associations (Dallinga et al., 2002). Charlier and colleagues recruited young male volunteers from a Belgian andrology clinic, comparing 82 men with male factor infertility with 73 fertile men to evaluate the association of semen characteristics (sperm concentration, motility, and morphology) and serum and seminal plasma p,p′-DDE concentrations. Cases had significantly lower sperm concentration, motility, and percent normal morphology than controls, however, there was no difference in serum or seminal plasma p,p′-DDE concentration in a comparison of the two groups (Charlier & Foidart, 2005). Utilizing data from Icelandic male infertility clinic patients with male (n=25) and female (n=20) factor infertility, Magnusdottir and colleagues evaluated the association of p,p′-DDE in relation to semen parameters (semen volume, sperm count, concentration and motility) (Magnusdottir et al., 2005). No associations were found between plasma or seminal plasma p,p′-DDE and these outcomes.
The two remaining infertility/andrology clinic-based studies were based in the U.S. and India. Hauser and colleagues evaluated the association between serum p,p′-DDE and abnormal semen quality (sperm count, concentration, motility and morphology) in a cross-sectional study of 212 male partners of subfertile couples at the Massachusetts General Hospital Andrology Laboratory (Hauser et al., 2003 p’-DDE; Hauser et al., 2005). Serum p,p′-DDE was not associated with any of the semen parameters evaluated (Hauser et al., 2003 p’-DDE; Hauser et al., 2005). The final study evaluated DDT and DDT metabolites (p,p′-DDE, p,p′-DDD, p,p′-DDT, and total DDT) in the seminal fluid of 45 men with male factor infertility and 45 controls with female factor infertility recruited from the obstetrics and gynecology department in Lucknow, India (Pant et al., 2004). Cases had significantly lower sperm count and sperm motility than controls and concentrations of p,p′-DDE, p,p′-DDD, and total DDT were higher in the seminal fluid of men with male factor infertility compared with controls (Pant et al., 2004).
Overall, there is very little evidence that DDT or its metabolites (DDE, DDD) are associated with impaired spermatogenesis in adult populations. Three of nine studies suggested that p,p′-DDE may be associated, albeit modestly, with decreased sperm motility, however these association were not adjusted for potential confounding factors, namely age. The other seminal characteristics studied, semen volume, sperm count, sperm concentration and morphology, were, almost overwhelmingly, not associated with any of the DDT metabolites evaluated. Taken together, the weight of the evidence suggests that exposure to DDT during adulthood is not associated with spermatogenesis.
To date, seven studies have evaluated the association between PCBs and spermatogenesis (Table 4) (Charlier & Foidart, 2005; Dallinga et al., 2002; Hauser et al., 2003 p’-DDE; Magnusdottir et al., 2005; Pant et al., 2004; Richthoff et al., 2003; Rignell-Hydbom et al., 2004; Rozati et al., 2002; Toft et al., 2005). Five of these studies also examined DDT and/or its metabolites and were summarized in the previous section (Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function); the two remaining studies were conducted in Sweden and India (Richthoff et al., 2003; Rozati et al., 2002). In a presumed-fertile study population from Sweden, Richthoff and colleagues evaluated the relationship between serum PCBs and semen parameters in 305 military recruits 18–21 years of age (Richthoff et al., 2003). The Indian study was based in Andrha Pradesh and recruited male volunteers from an infertility clinic (Rozati et al., 2002). They were able to compare 21 infertile men with 32 controls with proven fertility, to evaluate the association between seminal PCB concentrations and semen volume, sperm count, motility, morphology, vitality, and head defects.
In the study of Swedish fisherman, serum PCB-153 concentration was weakly associated with motility (mean difference 9.9%; 95% CI: −1% − 21%) but was not associated with the other semen variables (volume, count, and concentration) (Rignell-Hydbom et al., 2004 p’-DDE and male reproductive function). Similarly in the study of Swedish military recruits, total PCB concentration was negatively, albeit weakly (r = −0.13, p-value = 0.02), correlated with sperm motility. However, total PCB concentration was not associated with semen volume, sperm concentration or sperm count (Richthoff et al., 2003). The geographic comparison of Greenland, Ukraine, Sweden and Poland, provided little evidence that PCB exposure was related to sperm motility and also provided further evidence that PCBs were not associated with sperm count, concentration or morphology (Toft et al., 2005).
The remaining studies of PCB and spermatogenesis were conducted in infertility clinic settings. In the Netherlands, Dallinga et al. reported no difference in PCB concentration (congeners 118, 138, 153, 180) between infertile and fertile participants (Dallinga et al., 2002). Among men in the “normal” semen quality group (n=31), the authors reported a negative, albeit weak, correlation between total PCB exposure and sperm count (r= −0.37, p-value = 0.04) and progressively motile sperm density (r= −0.41, p-value = 0.02) (Dallinga et al., 2002). In the study of Icelandic male infertility clinic patients, Magnusdottir and colleagues reported no associations between twelve PCB congeners, or their sum, and semen parameters (semen volume, sperm count, concentration and motility), when data from all 72 participants was evaluated (Magnusdottir et al., 2005). In a U.S.-based study, Hauser et al. reported that serum PCB-138 concentration was associated with decreased sperm motility (OR for less than 50% normal sperm motility: tertile 2 vs. 1 = 1.68; tertile 3 vs. 1 = 2.35; p-trend = 0.03) and decreased percent normal morphology (OR for less than 4% normal sperm morphology: tertile 2 vs. 1 = 1.36; tertile 3 vs. 1 = 2.53; p-trend = 0.04) (Hauser et al., 2003 p’-DDE). There was no evidence of an association between PCB 118, PCB 153, the sum of all PCB congeners measured, or PCBs classified as cytochrome P450 enzyme inducers and any of the semen parameters (Hauser et al., 2003 p’-DDE). In the infertility clinic-based study in India, seminal plasma PCB concentrations were inversely correlated with semen volume (r = −0.68, p-value < 0.001), motility (r = −0.48, p-value < 0.001) and vitality (r = −0.79, p-value < 0.001), but they were not associated with sperm count, morphology or head defects (Rozati et al., 2002).
Similar to the DDT studies, the studies examining PCB exposure provided little evidence of an association with impaired spermatogenesis in adult populations. Associations between PCB exposure and semen volume, sperm count, sperm concentration and sperm morphology were for the most part null. PCB exposure was inversely correlated with sperm motility in three of seven studies; however, caution needs to be used to interpret these few studies. Further studies of PCB exposure (congener-specific and mixtures) and sperm motility are necessary to evaluate the potential association.
Three studies mentioned previously evaluated organochlorine compounds in addition to DDT and PCBs (Dallinga et al., 2002; Magnusdottir et al., 2005; Pant et al., 2004). In the Icelandic male infertility clinic study metabolites of chlordane (nonachlor and oxychlordane) were not associated with semen volume, sperm count, concentration and motility (Magnusdottir et al., 2005). Toxaphene, a mixture of approximately 200 organochlorines, was not associated with any of the semen characteristics measured in the Icelandic study (Magnusdottir et al., 2005). HCB was not associated with semen parameters in either the Icelandic or The Netherlands infertility clinic studies (Dallinga et al., 2002; Magnusdottir et al., 2005). Isoforms of hexachlorocyclohexane (HCH), a commercial insecticide, were not associated with any of the semen characteristics evaluated in either the Icelandic or Indian infertility clinic studies (Magnusdottir et al., 2005; Pant et al., 2004).
Only one study to date has evaluated in-utero exposure to organochlorines and semen quality (Guo et al., 2000). This study of large-scale cooking oil contamination in Taiwan compared semen parameters in twelve men whose mothers consumed PCB and PCDF contaminated cooking oil while pregnant with 23 men born to mothers from the same region who did not consume contaminated cooking oil during pregnancy (Guo et al., 2000). The study reported increased percent abnormal sperm morphology and decreased motility in exposed, relative to unexposed, men (Guo et al., 2000). Semen volume and sperm concentration were similar between the two groups (Guo et al., 2000). Mocarelli and colleagues evaluated semen parameters in men exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the 1976 eruption in Seveso, Italy (Mocarelli et al., 2008). Specifically, semen parameters of men exposed to TCDD during early life (1–9 years old), puberty (10–17 years old) and adulthood (18–26 years) were compared with semen parameters of unexposed healthy male controls (Mocarelli et al., 2008). TCDD exposure during early life was associated with decreased sperm concentration (p-value = 0.025), and motility (p-value = 0.018). TCDD exposure during puberty and adult life was not associated with spermatogenesis (Mocarelli et al., 2008).
The summarized literature suggests regional variations of both semen quality and organochlorine exposure. A handful of studies support weak associations between exposure to PCB and/or DDT or its metabolites and decreased sperm motility. However, the overall weight of the evidence does not support an association between PCB or DDT exposure and impaired spermatogenesis. PCB and DDT exposures are often highly correlated making it difficult to disentangle their potentially independent effects on spermatogenesis. Further, there are so few studies of other organochlorine exposure (chlordane, HCB, HCB, toxaphene) that it is unclear whether a dose-response relationship exists between organochlorine exposure and spermatogenesis. Finally, based on two small studies (Guo et al., 2000; Mocarelli et al., 2008), there is some evidence that in-utero or early life exposure to organochlorines may impact sperm motility. Given the lack of research on endocrine-disrupting chemicals during critical periods of development, in-utero, early life and puberty, additional studies of spermatogenesis and organochlorine exposure, specifically DDT and PCB, are warranted.
Evidence indicates that TGCT may be more likely to be the result of maldevelopment than the result of an accumulation of mutations leading to uncontrolled mitosis and loss of the ability to induce apoptosis. The unusual age of incidence curve supports such a premise: for non-infantile, or type II (Mostofi & Sesterhenn, 2004), TGCTs, the incidence increases at the onset of puberty and peaks at age 30 years before declining thereafter (McGlynn & Cook, 2009). The incidence of TGCTs has been increasing over time in many developed countries of predominant Caucasian ethnicity (Chia et al., 2010). These increases are consistent with a birth cohort effect (Bray et al., 2006; Bray et al., 2006; McGlynn et al., 2003; Moller, 1989), thus it is conceivable that the increasing use of organochlorines as pesticides (e.g. DDT, chlordane, mirex, β-HCH), fungicides (e.g. HCB), and coolants and insulators (e.g. PCBs) may be implicated in the pathogenesis of this relatively rare malignancy. This is especially so given that these organic compounds are able to mimic hormones, inducing gene expression patterns important in urogenital development and homeostasis (Roy et al., 2009; Toppari, 2008).
To date, there have been seven publications from five studies which have tested for associations between blood measures of organochlorines and TGCT (Biggs et al., 2008; Cohn et al., 2010; Hardell et al., 2003; Hardell et al., 2004; McGlynn et al., 2009; McGlynn et al., 2008; Purdue et al., 2009). These publications include a Swedish study (Hardell et al., 2003; Hardell et al., 2004), the Adult Testicular Lifestyle And blood Specimen (ATLAS) Study (Biggs et al., 2008), the Servicemen’s Testicular Tumor Environmental and Endocrine Determinants (STEED) Study (McGlynn et al., 2009; McGlynn et al., 2008), and two nested case – control studies; one within the Norwegian Janus Serum Bank cohort (Purdue et al., 2009) and one within the CHDS (Cohn et al., 2010). This section will first review the four studies that have assessed organochlorine exposure in men who had been, or were subsequently, diagnosed with TGCT compared with unaffected controls (Biggs et al., 2008; Hardell et al., 2003; Hardell et al., 2004; McGlynn et al., 2009; McGlynn et al., 2008; Purdue et al., 2009). Second, the section will compare the two studies that have assessed the association between maternal organochlorine levels and TGCT development in male offspring (Cohn et al., 2010; Hardell et al., 2003; Hardell et al., 2004).
Four case-control studies have assessed DDT and/or DDE in relation to TGCT (Table 5). All results for DDT have been null while the p,p′-DDE results were indicative of a positive association in three of the four studies (Hardell et al., 2003; McGlynn et al., 2008; Purdue et al., 2009), although only in the STEED Study was the association statistically significant (ptrend=0.0002) and this effect appeared to be restricted to the highest quartile of exposure (OR>0.39μg/g lipid=1.71, 95%CI:1.23–2.38) (McGlynn et al., 2008). Although the Swedish study (Hardell et al., 2003) found an non-statistically significant OR of 1.7 between p,p′-DDE exposure at or above the median level of controls and TGCT and the ATLAS Study found no association (Biggs et al., 2008), both of these studies had lower median exposure levels of p,p′-DDE (Swedishcontrol subjects=98 ng/g lipid; ATLASall subjects=153 ng/g lipid), fewer numbers of cases and controls available for analysis (Table 5), and collected bloods at or after diagnosis, respectively, compared with the STEED Study (McGlynn et al., 2008) which had higher levels of p,p′-DDE (mediancontrol subjects=251 ng/g lipid), larger numbers of study subjects, and pre- diagnostic blood samples. These differences would have tempered the ability of the Swedish (Hardell et al., 2003) and ATLAS studies (Biggs et al., 2008) to detect statistically significant effects. The case-control study nested within the Norwegian Janus Serum Bank cohort (Purdue et al., 2009) detected very high levels of p,p′-DDE (mediancontrol subjects=1833 ng/g lipid) using pre-diagnostic blood samples. The investigators reported a borderline statistically significant association between higher exposure levels and TGCT (pWilcoxon signed-rank test=0.07), although the number of samples included was modest. Therefore, the two studies which analyzed pre-diagnostic serum samples provide evidence for an association between increasing p,p′-DDE exposure and TGCT (McGlynn et al., 2008; Purdue et al., 2009). In the STEED Study, DDT could only be detected in 20% of samples tested and this may have reduced the ability to have detected a similar association and/or the effect may only manifest itself with DDE exposure (McGlynn et al., 2008). This is because, while DDT is estrogenic (White et al., 1994), DDE is known to have anti-androgenic effects (Kelce et al., 1995), which is consistent with evidence showing associations between anomalies characterized by hormonal perturbations and TGCT (Looijenga et al., 2010).
Three studies have assessed PCB exposure, as individual congeners and as functional groups, in relation to TGCT (Table 6). The STEED Study (McGlynn et al., 2009) found consistent inverse associations between PCB congeners, PCB functional groups and TGCT. These findings are not supported by the results of the Swedish (Hardell et al., 2004) and Norwegian (Purdue et al., 2009) studies, although these studies had much smaller sample sizes and only the Norwegian study had pre-diagnostic samples, making strict comparisons difficult. Regardless, there appears little evidence to support the premise that PCB exposure could increase one’s risk of TGCT; the Norwegian study (Purdue et al., 2009) did observe associations between PCB congeners 99 and 167, and TGCT, although the magnitude of these effects coupled with the relatively small sample size and number of association tests conducted warrant a cautious interpretation. Given the numbers of subjects available for analysis, and the consistency and magnitude of the inverse associations detected in the STEED Study (McGlynn et al., 2009), it has to be considered that PCB exposure could decrease TGCT risk. Although animal experiments would indicate the opposite, and hence the reason the hypothesis was tested, much of the animal literature is based on extremely high-dose exposures and effects of organochlorines can differ markedly between species (Golden & Kimbrough, 2009). Alternatively, PCB exposure could be a proxy for another, as yet unknown, exposure or interaction of exposures, with the results presented being the product of residual confounding. This proposition may have further support given that PCBs and their groupings are deemed to have varying biological effects (e.g. estrogenic, anti-estrogenic) (Wolff et al., 1997; Wolff & Toniolo, 1995).
Cyclodienes, derived from hexachlorocyclopentadiene, that have been assessed in relation to TGCT include chlordane, heptachlor, dieldrin, and mirex. Chlordane and its derivatives (oxychlordane, trans-nonachlor, cis-nonachlor, MC6) have been assessed by the four previously mentioned studies for association with TGCT (Table 5), and only the ATLAS Study (Biggs et al., 2008) failed to find at least one statistically significant association with these compounds. Associations with nonachlor have been found by the Swedish study (cis-nonachlor) (Hardell et al., 2003) and the STEED Study (cis- and trans-nonachlor) (McGlynn et al., 2008), and were borderline statistically significant in the Norwegian study (trans-nonachlor) (Purdue et al., 2009). The significant trends of trans- and cis-nonachlor with TGCT in the STEED Study were driven by associations with the top quartile of exposure (ORtrans-nonachlor=1.46, 95%CI:1.07–2.00; ORcis- nonachlor=1.56, 95%CI:1.11–2.18); the Norwegian (Purdue et al., 2009) and Swedish (Hardell et al., 2003) studies were not adequately powered for ordinal analyses, while the ATLAS Study found no association with the top tertile of trans-nonachlor exposure (ORtrans-nonachlor=0.89, 95%CI:0.49–1.61). However, quantitated median levels of trans- nonachlor where lower in the ATLAS (Biggs et al., 2008) and, for that matter, the Swedish (Hardell et al., 2003) study populations (ATLASall subjects=10.5 ng/g lipid; Swedishcontrol subjects=7.9 ng/g lipid) when compared with the STEED Study (trans- nonachlorcontrol subjects=16.6 ng/g lipid) (McGlynn et al., 2008) and the Norwegian study (trans-nonachlorcontrol subjects=20.5 ng/g lipid) (Purdue et al., 2009). These latter two studies, which indicated evidence for associations between trans-nonachlor, cis-nonachlor, total chlordanes and TGCT, also have the advantage of having assessed pre-diagnostic serum samples (McGlynn et al., 2008; Purdue et al., 2009). Thus, overall it appears as though cis- and trans-nonachlor may be associated with TGCT. Conversely, there is little evidence that the chlordane metabolites oxychlordane and MC6 are associated with TGCT, as was also true for other hexachlorocyclopentadiene derivatives of heptachlor, dieldrin, and mirex (Table 5).
Gamma-hexachlorocyclohexane (γ-HCH) and a by-product of its production, β-HCH, have been tested for association with TGCT (Table 5). With regards to γ-HCH, no evidence for a link has been found. One of the two studies to assess β-HCH did find evidence for association with TGCT (ORper 10pg/g serum=5.54, 95%CI:1.65–18.56) (Biggs et al., 2008). However, the categorical analysis and the multiple statistical tests conducted temper the evidence for association. The Norwegian study also examined β-HCH, but found no evidence of an association with TGCT.
Maternal organochlorine exposure should be considered separately from case exposure, as the former metric is assumed to affect TGCT pathogenesis in the prenatal (placental route) and neonatal (breast feeding) periods, while the exposure time window of importance is unknown for the latter and could be neonatal, prenatal, adolescent and/or adult. Carcinoma in-situ (CIS), a precursor lesion of TGCT, is postulated to arise during embryogenesis (Sonne et al., 2009) and the current TGCT paradigm suggests that most adults with CIS will eventually progress to TGCT (von der Maase et al., 1986). However, this latter statement is based on very little evidence and epidemiologic evidence indicates that post-natal exposures also exert effects on TGCT development (Cook et al., 2009; Cook et al., 2010; Czene et al., 2002; Lerro et al., 2010).
Only two studies have assessed maternal organochlorines exposure in relation to TGCT (Tables 5 and and6)6) (Cohn et al., 2010; Hardell et al., 2003; Hardell et al., 2004). Differences between these studies are notable, however, providing for a tenuous comparison of results. The Swedish study (Hardell et al., 2003; Hardell et al., 2004) collected maternal bloods after the son was diagnosed with TGCT, thus on average 30 years after birth. Although many organochlorine compounds have half-lives that extend for decades, BMI, changes in body weight, subsequent pregnancies, and breast-feeding can all affect levels of stored organochlorines in women (Guo et al., 1997; Wolff et al., 2000; Zietz et al., 2008). Thus, the long interval between exposure and exposure assessment makes interpretation of results difficult. Using this design, the Swedish study found no association between maternal DDE exposure and TGCT development in male offspring (Table 5) (Hardell et al., 2004).
The CHDS study (Cohn et al., 2010) measured maternal organochlorine exposure in an optimal design: a cohort study collected maternal bloods during or shortly after (within 3 days) pregnancy with the index son. Even in large cohorts, like the CHDS, however, only a small number of TGCT cases accrue due to the relatively low incidence. Thus, the CHDS study was able to assess organochlorines in just 15 mothers of TGCT cases matched 3:1 to mothers of control subjects. The study found an inverse association between maternal DDE and TGCT, which is the opposite of what is indicated from TGCT case-control studies previously reviewed. The authors conclude that this discrepancy may be caused by the timing of blood collection (perinatal vs. adolescent/adult) from which the exposure was quantitated (Hardell et al., 2004). The study also found an association between the DDT:DDE ratio and conclude that this may represent either a slower metabolism of DDT to DDE or more recent exposure in mothers of TGCT cases compared with mothers of control subjects. Although provocative, these hypothesis-generating findings require further study.
Maternal PCB exposure in relation to TGCT has only been assessed by the Swedish study (Hardell et al., 2004) which reported consistent and strong associations between 19 of the 37 PCB congeners tested and TGCT (Table 6). A dose-response relationship was not investigated, presumably because of the small number of subjects available for analysis. These strong and consistent results are in the opposite direction to those previously reviewed in the case-control analysis of the STEED Study (McGlynn et al., 2009) but are, to some extent, in agreement with the general direction of non-statistically significant associations reported from the Norwegian study (Purdue et al., 2009). Thus the reason for the discrepancy is unlikely to be due to differences in the source (maternal/case) or timing (pre-/post-diagnosis) of blood collection. Putative explanations for the discrepancy include unidentified confounding effects in US and/or Scandinavian populations, and non-linear effects between PCB exposure and TGCT.
Current epidemiological evidence would infer that p,p′-DDE and chlordane, particularly cis- and trans-nonachlor, are positively associated with TGCT. These results require validation in future studies. The sum of evidence does not support an association between TGCT and DDT, oxychlordane, MC6, heptachlor, dieldrin, mirex, HCB, and HCH. PCBs have been inversely and positively associated with TGCT, thus the effect of PCBs requires further investigation.
The overall weight of the evidences suggests that DDT, DDT metabolites, and PCBs are not associated with cryptorchidism, hypospadias or male fertility. p,p′-DDE appears to be positively associated with TGCT, but its parent compound, DDT, does not appear to be associated. Whether a relationship exists between PCBs and TGCT is not clear. There is much less research on other organochlorines and facets of TDS; those which have been investigated have been done so by a single or few studies with occasional tenuous associations. A potential exception is the relationship between chlordane-related compounds and TGCT, thus further studies of cis- and trans-nonachlor may prove fruitful. If any of the organochlorines discussed herein are causative in their relation to urogenital disorders, they may play a lesser role in the future given the declining atmospheric (Rigét et al., 2010) and biologic concentrations (Hardell et al., 2010; Hopf et al., 2009) of these exposures. Conversely, there remain many other endocrine-disrupting chemicals, including additional organochlorines, that have yet to be assessed in relation to disorders associated with TDS, but which are of interest given the scientific relevance of such hypotheses to urogenital development (Toppari, 2008).
There are several limitations of the human data reviewed herein, including: (1) Organochlorine exposures have varied in space and time, thus null findings one population may not necessarily contradict an association reported from a population with a different level of exposure. (2) Mixtures of organochlorine compounds have only been assessed with regard to groupings based on predicted biological effects (Wolff et al., 1997; Wolff & Toniolo, 1995). An optimal strategy would be to classify groups based on toxicity to the specific cell-type(s)/tissue(s) implicated in the disorder, but such effects are unknown as are whether mixtures of endocrine-disrupting chemicals exert unique effects. Current organochlorine classification schemes do attempt to parse out estrogenic and androgenic effects, which are relevant to etiologic hypotheses of the disorders reviewed (Sharpe, 2003; Toppari, 2008). (3) The timing/source (maternal/case) of biospecimen collection with respect to diagnosis of disease is variable, with some studies able to attain pre-diagnostic samples, others near the time of diagnosis, and other still many years after diagnosis. This concern is somewhat assuaged by the long half-life of organochlorines (Bennett et al., 1974; Wolff et al., 1992; Wolff et al., 2000), although it is difficult to reconcile results from maternal and case studies, especially with regards to fertility and TGCT, given the potentially extended time window in which organochlorine exposures may exert an effect. (4) Many studies have suboptimal sample sizes which has reduced the ability to detect potential associations between exposure and disease. Each of these points may have contributed to some of the inconsistencies of association across studies and should be kept in mind when critiquing the literature as a whole. Future studies should aim to assess exposures at the time window when they are considered to exert an effect, this is especially true for studies of congenital anomalies (prenatal) and fertility (prenatal and childhood/adolescence). Associations between an endocrine-disrupting chemical and disorder must be strong and consistent before mechanistic studies are warranted.
FUNDING; Intramural Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We would like to thank Ms. Pamela Lotinsky of DCEG, NCI for her help with citations, and proofing of the manuscript.
There are no financial disclosures from any of the authors.