Phthalate Monoesters and Infant Reproductive Health

In recent decades, reproductive health in adult men has revealed adverse trends in some, but not all, Western countries. The prevalence of testicular cancer is increasing and semen quality appears to decrease. However, there are large geographical variations in this phenomenon, the etiology of which is yet unknown [1]. Denmark and Finland, two Nordic countries with close vicinity and comparable modern life style, show a distinct difference in male reproductive health, which is better in Finland than in Denmark for all parameters.

It has recently been shown that a similar difference between these two Nordic countries exists for the prevalence of congenital malformations in boys, such as hypospadias and cryptorchidism, which are much more frequent in Denmark than in Finland [2] [3] [4]. 1.03% Danish boys had hypospadias at birth versus 0.27% Finnish boys. Congenital cryptorchidism was found in 9% and 1.9% of Danish boys at birth and 3 months of age, respectively, as opposed to 2.4% and 1.0% Finnish boys. The Danish prevalence of cryptorchidism had increased several-fold since a comparable cohort study in the same region of Denmark between 1959 and 1961, in which 1.8% of boys with normal birth weight showed congenital cryptorchidism. Even in healthy boys without visible genital malformations, there was a significant geographical difference in genital development. The Danish boys had smaller testes, less testicular growth during early infancy and a lower concentration of serum inhibin B, a marker for the size of the Sertoli cell pool and its function [5].

The prevalence of reproductive health problems is increasing rapidly. Therefore, genetic differences alone cannot account for the findings, but lifestyle and environmental factors need to be taken into consideration as well [6]. Toxicological studies have revealed endocrine-disrupting activities of numerous man-made chemicals that are found in the environment with adverse effects on testicular or ovarian function, however, often at doses irrelevant to human exposure levels (www.atsdr.cdc.gov/toxprofiles). Some of these chemicals have been banned from use in Western countries, but may still be found in the environment due to slow biodegradation and bioaccumulation via the food chain. Other chemicals, such as plasticizers (phthalates), are still produced in large and even increasing amounts. Phthalates are used in a wide range of products to induce flexibility, preserve colour and odour, i.e. in PVC flooring, cosmetics, toys, food wrapping and medical equipment [7]. Thus, despite their rapid metabolism, humans are exposed continuously and through many sources to phthalate diesters by ingestion, inhalation or dermal contact [8]. In agreement with this, phthalate metabolites have been detected in human matrices such as breast milk, serum, urine, amniotic fluid, saliva and seminal fluid. Due to a larger ingestion of calories per kilogram, a larger surface to volume ratio and oral exploratory behavior, children, especially young infants, appear to have a higher general exposure than adults [9].

The development of the human reproductive tract in boys is highly dependent on an intact hormonal balance during pregnancy and infancy. A disruption of testicular development during this phase in life may compromise testicular function permanently. In 2001, a hypothesis was put forward, that adult male reproductive problems were closely linked to congenital malformations when reviewing the available evidence of epidemiological studies, histological and genetic markers of fetal and adult cell function and looking at common risk factors for all entities [10]. Any perinatal disturbance in testicular development can lead to signs of testicular dysgenesis such as testis cancer, reduced semen quality, cryptorchidism and hypospadias. The recent Danish-Finnish birth cohort study supports this hypothesis of a prenatal onset of testicular dysfunction [11].

In 1997, a large prospective baby cohort study was launched in Denmark and Finland in the attempt to find risk factors for cryptorchidism, which were associated with life style and environmental exposure by means of a comprehensive antenatal questionnaire, repetitive standardized clinical examination of the children and a bio bank with samples from mother and child [2] [12]. At three months of age infant boys have a brief activation of the hypothalamus-pituitary-gonadal axis, a feature, which can be applied as a diagnostic ‘window’ of testicular function [13]. Thus, in boys with congenital cryptorchidism, subtle elevations in gonadotropins level could be detected as a sign of impaired primary testicular function [14].

Breast milk samples from 130 mothers of 62 cryptorchid and 68 healthy boys, collected 1 to 3 months post partum, were selected for the analysis of six most common phthalate metabolites: mMP (mono-methyl phthalate), mEP (mono-ethyl phthalate), mBP (mono-butyl phthalate), mBzP (mono-benzyl phthalate), mEHP (mono-ethylhexyl phthalate) and miNP (mono-isononyl phthalate) and compared to serum levels of reproductive hormones at three months of age in the infant boys [15] [12]. All phthalate metabolites were detected in the breast milk samples at very different absolute values, with mMP having the lowest and miNP the highest concentrations ([Table 1]). There was a considerable variation in the concentration of individual samples. The concentrations of mMP, mEP, mBP, mBzP and mEHP were positively correlated to each other (r=0.24-0.43, p=0.0001), indicating a common source of exposure. No association between phthalate concentration in breast milk and cryptorchidism was found, but there was a correlation between the concentration of some phthalate monoesters and the ratio of LH/free testosterone (mMP, mEP, mBP), SHBG (mEP, mBP), free testosterone (mBP) and LH (miNP) in infant serum. A 10-fold increase in mEP or mBP raised serum SHBG levels by 15 and 8%, respectively. A 10-fold increase in mMP, mEP or mBP concentrations raised the LH/free testosterone ratio by mean 19, 26 or 18%, respectively. Free testosterone decreased with 15% over a 10-fold increase of mBP. A 10-fold increase of miNP raised LH levels by 97%. The findings indicated a decreased androgen bioactivity and suggested a subtle effect of phthalate exposure on human Leydig cell function.

Table 1 Concentrations (medians, range in parenthesis) of six phthalate monoesters in human breast milk samples collected 1997-2001 from 65 Danish and 65 Finnish mothers Phthalate monoester μg/L Denmark N=65 Finland N=65 *significant country difference p<0.001 MMP 0.10 (<0.01-5.53) 0.09 (<0.01-0.37) MEP 0.93 (0.07-33.6) 0.97 (0.25-41.4) MBP* 4.3 (0.6-10900) 12 (2.4-123) MBzP* 0.9 (0.2-14) 1.3 (0.4-26) MEHP* 9.5 (1.5-191) 13 (4.0-1410) MiNP 101 (27-469) 89 (28-230)

In another infant study from the US, anti-androgenic effects of phthalate exposure were also seen. In this study phthalate exposure was assessed antenatally by measurement of metabolites in maternal urine. Infants were examined postnatally for cryptorchidism and genital development, including a new biomarker of toxic reproductive effect: the anogenital distance. This distance was measured between the center of the anus and the anterior base of the penis. It was adjusted for infant body weight to correct for postnatal growth [16]. In rodents and humans, anogenital distance is gender dimorphic, and in rodents antiandrogenic effects result in a reduction of the anogenital distance. The higher the concentration of four phthalate metabolites in maternal urine (mEP, mBP, MBzP and mono-isobutyl phthalate miBP), the shorter the anogenital distance in her male offspring. Boys with a short anogenital distance had a higher prevalence of cryptorchidism and a smaller penile size. These parameters taken together were indicative of an anti-androgenic effect.

In conclusion, the anti-androgenic effect of in utero phthalate exposure, which has been well documented in rodents for especially mBP and mEHP, has for the first time also been described in two human studies of infant boys. The changes observed in humans were more subtle and occurred at lower (environmental) exposure doses than usually applied in rodent experiments. It cannot be excluded, that simultaneous exposure to other chemicals than phthalates may contribute to the observed findings, as infants are exposed to more than one chemical at a time [17] [18]. In addition, lifestyle factors may contribute to the overall risk [4] [19]. However, the studies suggest that human testicular development pre-and perinatally may be vulnerable to phthalate exposure. Breast milk appeared in this context to be a suitable matrix to monitor infant exposure to phthalates, similar to measurement of urinary phthalate excretion.

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