An Update on Phthalates and Male Reproductive Development and Function
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- Grady, R. & Sathyanarayana, S. Curr Urol Rep (2012) 13: 307. doi:10.1007/s11934-012-0261-1
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Phthalates are part of a class of high-volume industrial chemicals used ubiquitously in health care as well as household products. Biomarker studies have confirmed the widespread presence of these chemicals in both humans and animals. As a class, phthalates have been implicated in diseases and birth anomalies of the genitourinary tract including hypospadias, testis anomalies, and subfertility. This article will discuss the current data surrounding these chemicals and their association with genital anomalies including genital anomalies and infertility.
KeywordsPhthalatesAnogenital distanceInfertilityHypospadiasCryptorchismEndocrine disruptorBirth anomaliesGenitourinary tractTestis anomaliesSubfertility
Phthalates comprise a class of environmentally pervasive chemicals; many of them appear to have antiandrogenic activity. This class of industrial chemicals is used in a broad variety of applications and is among the most abundant synthetic chemicals in the environment. Phthalates recently have become subjected to increased scrutiny because of a growing body of evidence in animals implicating them in a spectrum of reproductive disorders. Recent human data also have shown results consistent with these findings. According to the Centers for Disease Control and Prevention (CDC), nearly 100 % of the U.S. population exhibits measurable levels of one or more of these chemicals . Phthalate esters are high–production volume chemicals used to impart flexibility to plastics as well as many other applications. High–molecular weight phthalates, such as di (2-ethylhexyl) phthalate (DEHP), are primarily used as plasticizers in the manufacture of polyvinyl chloride. Phthalate esters are found in many commonly used products, including children’s toys, health and beauty supplies (eg, cosmetics and perfumes), medical equipment (eg, dialysis tubing and intravenous bags), and the enteric coating of some pharmaceuticals [2, 3]. Phthalates have been measured in residential indoor environments in both house dust and indoor air . They also have been measured in foods, milk, and drinking water. However, the relative contribution from the various sources and routes of exposure to phthalates is unknown . Phthalate esters readily migrate from such products, and their metabolites have been detected in several human bodily fluids, including maternal urine during pregnancy , breast milk , and amniotic fluid .
Some phthalates reduce the production of androgens by the testis. Over the past 7 years, increasingly detailed rodent studies have demonstrated that several phthalates (most notably DEHP [9, 10] and di-n-butyl phthalate [DBP] [11, 12]), when administered at the critical window for the development of the reproductive tract, disrupt the androgen-signaling pathway. The androgen-signaling pathway is disrupted by phthalate esters via alterations of testis cell hormone production (and insulin-like peptide 3 [insl3] is downregulated as well). These phthalates can significantly reduce fetal testicular testosterone production during the critical window of sex differentiation (a time when androgen production by the fetus is not under luteinizing hormone (LH) control, nor does the male fetus get androgen from the dam).
In rodents exposed to phthalates, a syndrome of genital dysmorphology has been identified and named the “phthalate syndrome” . This syndrome includes incomplete testicular descent, smaller testis weight, and penile size as well as alterations to the vas deferens and epididymis, and most notably, shortened anogenital distance (AGD), a sexually dimorphic phenotypic biomarker. While not all of these outcomes are measurable in a clinical setting, alterations in penile width and testicular location are found to be related to DEHP metabolites . Recent work has clearly differentiated the phthalate syndrome from the effects other antiandrogens, which, unlike phthalates, are androgen-receptor (AR) agonists. In addition to inhibiting testosterone synthesis, the phthalates DEHP and DBP also reduce expression of insl3, an important hormone secreted by the Leydig cell and necessary for development of the gubernacular ligament, which results in incomplete decent of the testis. This reduction of insl3 by phthalates is not caused by antiandrogens that are AR agonists (eg, vinclozolin and procymidone) . The adverse outcomes on male reproductive development following in utero phthalate exposure affect Wolffian duct and prostate differentiation, along with inducing hypospadias and cryptorchidism.
Deterioration in semen quality has been reported in both animal and human populations. A recent meta-analysis of semen quality demonstrated declines in sperm concentration in Europe and North America between 1934 and 1996 . It is difficult to determine if similar trends might be occurring in domestic animal populations because animal husbandry practices such as culling of the reproductively inefficient food- and fiber-producing animals at an early age contaminate otherwise naturally occurring trends. Phthalate exposure has been associated with decreased male fertility, specifically decreased semen quality in human exposure studies [16, 17]. Animal studies have also linked DEHP exposure to decreased fertility .
Animal Studies/Basic Research
Recently Lee et al.  demonstrated the anti-androgenic effects of seven phthalates (DEHP and its metabolite, mono-2-ethyhexyl phthalate [MEHP], DBP, benzylbutylphthalate [BBzP] , as well as di-isononyl phthalate [DINP], di-isodecyl phthalate [DIDP], di-n-heptyl phthalate [DnHP]) by the Hershberger assay in castrated male SD rats. This antiandrogenic action results in interference with the normal cascade of androgen-dependent outcomes, most notably shortened AGD. Several animal studies report that DEHP and DBP induce a marked reduction in fetal testosterone (produced by Leydig cells) and insulin-like growth factor-3 (IGF-3), resulting in a syndrome of male reproductive abnormalities, which, in addition to shortened anogenital distance (AGD), include hypospadias, cryptorchidism and malformations of the epididymis, vas deferens, seminal vesicles, and prostate, together comprising the “phthalate syndrome” . While a variety of antiandrogens alter AGD, most are AR antagonists. The syndrome induced by the AR antagonists differs from that induced by the phthalates, which inhibit the synthesis of fetal testosterone and Insl-3 .
AGD is a sexually dimorphic trait that develops in utero; the growth of the perineum in utero is under direct androgen control. Hence in men, AGD at birth is about double, on average, that for females due to the significantly higher androgen levels (in rats these fetal levels of testosterone [T] reach almost adult values). In the fetal testis, T is converted to DHT and this potent androgen stimulates growth of the tissue.
Shortened male AGD also has been reported following exposure to a number of endocrine-disrupting chemicals (EDCs) including phthalates. Gray et al.  tested a variety of potentially antiandrogenic substances including procymidone (P), linuron (L), flutamide (F), and p,p’-DDE (D) and found that P, L, and D produced F-like profiles that included shortened AGD, but are distinct from those produced by DBP and DEHP, and concluded that D, P, and L alter male sexual differentiation via different mechanisms. Thus, detection of shortened AGD appears to signal prenatal antiandrogen exposure, but knowledge of the entire profile of genital dysmorphology is necessary to pinpoint a particular etiologic agent. In utero exposure to these phthalates induced a low incidence of abnormalities consistent with the “phthalate syndrome” along with subtle reductions in reproductive organ weights. This includes hypospadias, an arrest in normal development of the urethra, foreskin, and ventral aspect of the penis, and impaired testicular descent (cryptorchidism) as well as other genital malformations not externally visible. In the high-dose group, more than 25 % of the men displayed testicular and/or epididymal abnormalities .
Pant and his coworkers  evaluated the effect of phthalates on human sperm. To conduct the experiment, phthalate esters were analyzed by high-performance liquid chromatography and cell viability was assessed by MTT assay. The studies were performed in vitro with specimens exposed to the highest phthalate concentration in semen samples found in vivo for a period ranging between 30 min and 96 h. An inverse relationship with sperm motility was noted with significant dose-and time-dependent decrease in the sperm motility under in vitro environment after 12-hour exposure. Cytotoxicity was observed at the highest concentration of phthalates after 96 h of exposure. The authors also found a significant correlation between phthalate ester DEHP, DBP, and sperm motility both in vitro and in vivo. The mechanism for this effect was not explored; the study does suggest a basis for epidemiologic studies in the literature that associate decreased fertility with phthalate exposure . Basic research and animal studies exploring prenatal exposure of phthalates to later fertility effects remain to be done.
Human exposure studies documenting the presence of DEHP and DBP and other phthalates in human tissue have established an accepted consistent presence of these chemicals in human systems . Only a few studies have examined the effects of pre- and perinatal exposure to these phthalates on the development of the genitourinary system in humans. The Phthalates in Pregnant Women and Children (PPWC) was one of the first studies to examine this association [6, 26]. In this study, nine phthalate monoester metabolites were measured in urine samples provided by mothers during pregnancy as predictors of (age- and weight-adjusted) AGD. When a mother’s concentration of any of five phthalate metabolites (including metabolites of DEHP and DBP) was “high” (at 75th percentile or higher), the odds of her son having a short AGD (less than 25 % expected for his age and weight) was increased between 3 and 29 times, compared to sons of mothers whose exposure was “low” (below the 25th percentile). The decrease in age- and weight-adjusted AGD was even greater when the mother was exposed to high levels of all or most of these metabolites, consistent with the dose additively found in rodent studies . The “high” concentrations associated with these decreases in AGD were well within current environmental levels.
Huang and colleagues  examined the relationship of AGD (and AGD/weight) in 33 male newborns to DEHP and DBP metabolites in amniotic fluid and urine collected on the same day. Exposure was dichotomized at the median. No association was seen between these metabolites and male AGD, although a shorter AGD was noted in female offspring of mothers with higher concentrations of mono butyl phthalate (MBP), which has not been observed in rodent studies . No other studies have examined reproductive development following prenatal phthalate exposure. The implications of shortened male AGD are unclear and require further investigation. However, clinical studies linking AGD to infertility will be discussed later in this article.
The PPWC study was the only study to examine AGD in relation to penile size and testicular descent in humans. As predicted by animal data, these androgen-sensitive measures were correlated. Testicular descent in relation to prenatal phthalate exposure also has been examined. Among boys whose mothers had higher levels of any of three DEHP metabolites, 24 % had incompletely descended testis as defined by one or more not “normal” or “normal retractile” compared to none of the boys whose mothers had low concentration of these metabolites. A decrease in penile width was associated with these exposures; however, no cases of hypospadias were seen, likely due to the small sample size of the population (n = 106) .
While no other study looked at perinatal phthalate exposure in relation to genital measurements, hormonal concentrations at specific developmental periods may affect the development of these outcomes. Main and colleagues  found that the phthalate metabolite MBP in breast milk was negatively correlated with the infant’s free testosterone (r = −0.43, P = 0.004) and positively with LH/free testosterone (r = 0.46, P = 0.001).
The effect of phthalates on human male fertility is currently being approached in two directions. Several human studies have shown that phthalates are associated with a direct adverse effect on androgen function in men. Calafat and her coworkers  found an inverse relationship with circulating steroid hormone levels (testosterone, estradiol, and free androgen index) and MEHP. These results suggest a potential mechanism through aromatase suppression. Urinary phthalate monoester concentrations also have been shown to be higher in couples with infertility problems compared to couples with children .
Recognizing that AGD length is affected by phthalate exposure in utero in both animal and human studies , several studies have explored the relationship between AGD and fertility in humans in an effort to study the testicular dysgenesis syndrome hypothesis. In one provocative study, shortened AGD has been associated with decreased fertility. Mendiola and colleagues [32••] recruited 126 adult male volunteers and performed AGD measurements along with semen analyses and found that anoscrotal distance [AGD(AS)] was associated with decreased semen parameters (sperm concentration, motility, morphology, total sperm count, and total motile count [P values 0.002–0.048]). In addition, men with AGD(AS) below the median were 7.3 times more likely to have a low sperm concentration (<20 × 10(6)/mL). A recently published study by Eisenberg and colleagues [33••] also demonstrated a relationship between AGD and improved semen parameters after varicocele repair. In this study, shortened AGD was associated with significantly less improvement in semen quality for men with a varicocele and shortened AGD. Specifically, 48 % of men with a shorter AGD had improvements in sperm concentration postoperatively compared to 84 % of men with a longer AGD improved. The authors also noted a trend toward a lower percentage of men (62 % vs 84 %), with shorter AGDs showing improvements in total motile sperm count. These results suggest that early exposure to endocrine-disrupting chemicals like phthalates that have been shown to adversely affect AGD may have implications for later male reproductive health.
Phthalates are a class of industrial chemicals that are ubiquitously found in the environment. They have been shown to have endocrine-disrupting effects at levels that are found in the environment and in vivo. Animal and human studies suggest that these chemicals may be impacting the development and function of the male reproductive system.
No potential conflicts of interest relevant to this article were reported.