Helena E. Virtanen

Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age.

Phthalates adversely affect the male reproductive system in animals. We investigated whether phthalate monoester contamination of human breast milk had any influence on the postnatal surge of reproductive hormones in newborn boys as a sign of testicular dysgenesis. Design We obtained biologic samples from a prospective Danish–Finnish cohort study on cryptorchidism from 1997 to 2001. We analyzed individual breast milk samples collected as additive aliquots 1–3 months postnatally (n = 130; 62 cryptorchid/68 healthy boys) for phthalate monoesters [mono-methyl phthalate (mMP), mono-ethyl phthalate (mEP), mono-n-butyl phthalate (mBP), mono-benzyl phthalate (mBzP), mono-2-ethylhexyl phthalate (mEHP), mono-isononyl phthalate (miNP)]. We analyzed serum samples (obtained in 74% of all boys) for gonadotropins, sex-hormone binding globulin (SHBG), testosterone, and inhibin B. Results All phthalate monoesters were found in breast milk with large variations [medians (minimum–maximum)]: mMP 0.10 (< 0.01–5.53 μg/L), mEP 0.95 (0.07–41.4 μg/L), mBP 9.6 (0.6–10,900 μg/L), mBzP 1.2 (0.2–26 μg/L), mEHP 11 (1.5–1,410 μg/L), miNP 95 (27–469 μg/L). Finnish breast milk had higher concentrations of mBP, mBzP, mEHP, and Danish breast milk had higher values for miNP (p = 0.0001–0.056). No association was found between phthalate monoester levels and cryptorchidism. However, mEP and mBP showed positive correlations with SHBG (r = 0.323, p = 0.002 and r = 0.272, p = 0.01, respectively); mMP, mEP, and mBP with LH:free testosterone ratio (r = 0.21–0.323, p = 0.002–0.044) and miNP with luteinizing hormone (r = 0.243, p = 0.019). mBP was negatively correlated with free testosterone (r = −0.22, p = 0.033). Other phthalate monoesters showed similar but nonsignificant tendencies. Conclusions Our data on reproductive hormone profiles and phthalate exposures in newborn boys are in accordance with rodent data and suggest that human Leydig cell development and function may also be vulnerable to perinatal exposure to some phthalates. Our findings are also in line with other recent human data showing incomplete virilization in infant boys exposed to phthalates prenatally.

Difference in prevalence of congenital cryptorchidism in infants between two Nordic countries.

BACKGROUND Several investigators have shown striking differences in semen quality and testicular cancer rate between Denmark and Finland. Since maldescent of the testis is a shared risk factor for these conditions we undertook a joint prospective study for the prevalence of congenital cryptorchidism. METHODS 1068 Danish (1997-2001) and 1494 Finnish boys (1997-99) were consecutively recruited prenatally. We also established prevalence data for all newborns at Turku University Central Hospital, Finland (1997-99, n=5798). Testicular position was assessed by a standardised technique. All subtypes of congenital cryptorchidism were included, but retractile testes were considered normal. FINDINGS Prevalence of cryptorchidism at birth was 9.0% (95% CI 7.3-10.8) in Denmark and 2.4% (1.7-3.3) in Finland. At 3 months of age, prevalence rates were 1.9% (1.2-3.0) and 1.0% (0.5-1.7), respectively. Significant geographic differences were still present after adjustment for confounding factors (birthweight, gestational age, being small for gestational age, maternal age, parity, mode of delivery); odds ratio (Denmark vs Finland) was 4.4 (2.9-6.7, p<0.0001) at birth and 2.2 (1.0-4.5, p=0.039) at three months. The rate in Denmark was significantly higher than that reported 40 years ago. INTERPRETATION Our findings of increasing and much higher prevalence of congenital cryptorchidism in Denmark than in Finland contribute evidence to the pattern of high frequency of reproductive problems such as testicular cancer and impaired semen quality in Danish men. Although genetic factors could account for the geographic difference, the increase in reproductive health problems in Denmark is more likely explained by environmental factors, including endocrine disrupters and lifestyle.

Flame retardants in placenta and breast milk and cryptorchidism in newborn boys

Background Polybrominated diphenyl ethers (PBDEs) are widely used in Western countries. Objectives Because the prevalence of cryptorchidism appears to be increasing, we investigated whether exposure to PBDEs was associated with testicular maldescent. Methods In a prospective Danish–Finnish study, 1997–2001, all boys were examined for cryptorchidism. We analyzed whole placentas (for 95 cryptorchid/185 healthy boys) and individual breast milk samples (62/68) for 14 PBDEs and infant serum samples for gonadotropins, sex-hormone binding globulin, testosterone, and inhibin B. Results In 86 placenta–milk pairs, placenta PBDE concentrations in fat were lower than in breast milk, and a larger number of congeners were nondetectable. There was no significant difference between boys with and without cryptorchidism for individual congeners, the sum of 5 most prevalent, or all 14 congeners. The concentration of PBDEs in breast milk was significantly higher in boys with cryptorchidism than in controls (sum of BDEs 47, 153, 99, 100, 28, 66, and 154: median, 4.16 vs. 3.16 ng/g fat; p < 0.007). There was a positive correlation between the sum of PBDEs and serum luteinizing hormone (p < 0.033). The sum of PBDEs in breast milk did not differ between Denmark and Finland (median, 3.52 vs. 3.44 ng/g fat), but significant differences in some individual congeners were found. Conclusions Two different proxies were used for prenatal PBDE exposure, and levels in breast milk, but not in placenta, showed an association with congenital cryptorchidism. Other environmental factors may contribute to cryptorchidism. Our observations are of concern because human exposure to PBDEs is high in some geographic areas.

Persistent Pesticides in Human Breast Milk and Cryptorchidism

Introduction Prenatal exposure to some pesticides can adversely affect male reproductive health in animals. We investigated a possible human association between maternal exposure to 27 organochlorine compounds used as pesticides and cryptorchidism among male children. Design Within a prospective birth cohort, we performed a case–control study; 62 milk samples from mothers of cryptorchid boys and 68 from mothers of healthy boys were selected. Milk was collected as individual pools between 1 and 3 months postpartum and analyzed for 27 organochlorine pesticides. Results Eight organochlorine pesticides were measurable in all samples (medians; nanograms per gram lipid) for cases/controls: 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p′-DDE): 97.3/83.8; β-hexachlorocyclohexane (β-HCH): 13.6/12.3; hexachlorobenzene (HCB): 10.6/8.8; α -endosulfan: 7.0/6.7; oxychlordane: 4.5/4.1; 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p′-DDT): 4.6/4.0; dieldrin: 4.1/3.1; cis-heptachloroepoxide (cis-HE): 2.5/2.2. Five compounds [octachlorostyrene (OCS); pentachlorobenzene, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p′-DDD); o,p′-DDT; mirex] were measurable in most samples (detection rates 90.8–99.2%) but in lower concentrations. For methoxychlor, cis-chlordane, pentachloroanisole (PCA), γ -HCH, 1,1-dichloro-2-(2-chlorophenyl)-2,2(4-chlorophenyl)ethane, trans-chlordane, α -HCH, and o,p′-DDE, both concentrations and detection rates were low (26.5–71.5%). Heptachlor, HCH (δ, ɛ ), aldrin, β-endosulfan and trans-heptachloroepoxide were detected at negligible concentrations and low detection rates and were not analyzed further. Seventeen of 21 organochlorine pesticides [p,p′-DDT, p,p′-DDE, p,p′-DDD, o,p′-DDT, HCH (α , β, γ ), HCB, PCA, α -endosulfan, cis-HE, chlordane (cis-, trans-) oxychlordane, methoxychlor, OCS, and dieldrin] were measured in higher median concentrations in case milk than in control milk. Apart from trans-chlordane (p = 0.012), there were no significant differences between cryptorchid and healthy boys for individual chemicals. However, combined statistical analysis of the eight most abundant persistent pesticides showed that pesticide levels in breast milk were significantly higher in boys with cryptorchidism (p = 0.032). Conclusion The association between congenital cryptorchidism and some persistent pesticides in breast milk as a proxy for maternal exposure suggests that testicular descent in the fetus may be adversely affected.

Nordic consensus on treatment of undescended testes

Aim: To reach consensus among specialists from the Nordic countries on the present state‐of‐the‐art in treatment of undescended testicles.

Cryptorchidism: classification, prevalence and long-term consequences

Undescended testis is a common finding in boys, and the majority of cases have no discernible aetiology. There are unexplained geographical differences and temporal trends in its prevalence. Cryptorchidism, especially bilateral, is associated with impaired spermatogenesis and endocrine function and increases the risk of testicular cancer. There is an urgent need to identify factors that adversely affect testicular development and optimize treatment.

Cryptorchidism and hypospadias as a sign of testicular dysgenesis syndrome (TDS): Environmental connection

Cryptorchidism and hypospadias are common genital birth defects that affect 2-9% and 0.2-1% of male newborns, respectively. The incidence of both defects shows large geographic variation, and in several countries increasing trends have been reported. The conditions share many risk factors, and they are also interlinked to the risk of testis cancer and poor semen quality. Testicular Dysgenesis Syndrome (TDS) may underlie many cases of all these male reproductive health problems. Genetic defects in androgen production or action can cause both cryptorchidism and hypospadias, but these are not common. A monogenic reason for cryptorchidism or hypospadias has been identified only in a small proportion of all cases. Environmental effects appear to play a major role in TDS. Exposure to several persistent chemicals has been found to be associated with the risk of cryptorchidism, and exposure to anti-androgenic phthalates has been shown to be associated with hormonal changes predisposing to male reproductive problems. Despite progress in identification of endocrine-disrupting substances, we are still far from knowing all the risk factors for these birth defects, and advice for prevention must be based on precautionary principles.

Recent adverse trends in semen quality and testis cancer incidence among Finnish men

Impaired semen quality and testicular cancer may be linked through a testicular dysgenesis syndrome of foetal origin. The incidence of testis cancer has been shown to increase among Finnish men, whereas there is no recent publication describing temporal trends in semen quality. Therefore, we carried out a prospective semen quality study and a registry study of testis cancer incidence among Finnish men to explore recent trends. A total of 858 men were investigated in the semen quality study during 1998–2006. Median sperm concentrations were 67 (95% CI 57–80) million/mL, 60 (51–71) and 48 (39–60) for birth cohorts 1979–81, 1982–83 and 1987; total sperm counts 227 (189–272) million, 202 (170–240) and 165 (132–207); total number of morphologically normal spermatozoa 18 (14–23) million, 15 (12–19) and 11 (8–15). Men aged 10–59 years at the time of diagnosis with testicular cancer during 1954–2008 were included in the registry study, which confirmed the increasing incidence of testicular cancer in recent cohorts. These simultaneous and rapidly occurring adverse trends suggest that the underlying causes are environmental and, as such, preventable. Our findings necessitate not only further surveillance of male reproductive health but also research to detect and remove the underlying factors.

A genome-wide association study of men with symptoms of testicular dysgenesis syndrome and its network biology interpretation

Background Testicular dysgenesis syndrome (TDS) is a common disease that links testicular germ cell cancer, cryptorchidism and some cases of hypospadias and male infertility with impaired development of the testis. The incidence of these disorders has increased over the last few decades, and testicular cancer now affects 1% of the Danish and Norwegian male population. Methods To identify genetic variants that span the four TDS phenotypes, the authors performed a genome-wide association study (GWAS) using Affymetrix Human SNP Array 6.0 to screen 488 patients with symptoms of TDS and 439 selected controls with excellent reproductive health. Furthermore, they developed a novel integrative method that combines GWAS data with other TDS-relevant data types and identified additional TDS markers. The most significant findings were replicated in an independent cohort of 671 Nordic men. Results Markers located in the region of TGFBR3 and BMP7 showed association with all TDS phenotypes in both the discovery and replication cohorts. An immunohistochemistry investigation confirmed the presence of transforming growth factor β receptor type III (TGFBR3) in peritubular and Leydig cells, in both fetal and adult testis. Single-nucleotide polymorphisms in the KITLG gene showed significant associations, but only with testicular cancer. Conclusions The association of single-nucleotide polymorphisms in the TGFBR3 and BMP7 genes, which belong to the transforming growth factor β signalling pathway, suggests a role for this pathway in the pathogenesis of TDS. Integrating data from multiple layers can highlight findings in GWAS that are biologically relevant despite having border significance at currently accepted statistical levels.

A Common Polymorphism in the Human Relaxin-Like Factor (RLF) Gene: No Relationship with Cryptorchidism

The incidence of impaired testicular descent (cryptorchidism) is high; 1–2% of boys at the age of 3 mo are diagnosed for this condition in western countries. Recent data on mice with targeted disruption of the Insl3/relaxin-like factor (RLF) gene proposed that this factor plays a role in testicular descent in fetal life. Male RLF−/− mice exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, associated with abnormal spermatogenesis and infertility. In the present study, we have sequenced the promoter region and both exons of the RLF gene in a cohort of 30 boys, seven of whom presented with a possible familial form of cryptorchidism and 23 with sporadic cryptorchidism. One of the nucleotide substitutions detected, G to A at position 178, predicted amino acid change. The mutation was localized to the C-peptide region, resulting in an alanine to threonine change and therefore classified as a conservative mutation. Four of the 30 cases analyzed were homozygous (13%), and 15 were heterozygous for the mutation (50%). However, the same mutation was also found in a control group of 89 men; 10% of them were homozygous, and 39% were heterozygous. Our results indicate that mutations in the RLF gene are not a common reason for cryptorchidism and that the common G178A polymorphism has no apparent relationship with this condition.