Marko Kaleva

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.

Function of stem cell factor as a survival factor of spermatogonia and localization of messenger ribonucleic acid in the rat seminiferous epithelium.

To address the possibility that stem cell factor (SCF) is a paracrine regulator of germ cell development in the adult rat testis, stage-specific distribution of SCF messenger RNA (mRNA) was investigated with Northern blot and in situ hybridization analyses. The highest levels of SCF mRNA were found in stages II–VI of the rat seminiferous epithelial cycle, whereas the lowest levels were in stages VII–VIII. Intermediate levels of SCF mRNA were detected in stages IX–XIV–I of the cycle. The expression of the SCF gene was found to be developmentally regulated, and the expression pattern followed the process of Sertoli cell proliferation and differentiation during postnatal life. The effect of mouse recombinant SCF on spermatogonial DNA synthesis was studied using an in vitro tissue culture system for stage-defined seminiferous tubules. A significant increase in DNA synthesis in spermatogonia could be detected when tubule segments from stage XII were cultured in the presence of 100 ng/ml SCF for 48 h (P < 0.05)...

Apoptotic Cell Death in the Normal and Cryptorchid Human Testis: The Effect of Human Chorionic Gonadotropin on Testicular Cell Survival

Cryptorchidism is associated with histologic changes in the human testis apparent by 2 y of age. The mechanism accounting for these changes is still unknown. To clarify whether apoptosis plays a role in human cryptorchidism, we evaluated its occurrence in cryptorchid testes of 73 prepubertal boys, 43 of whom had received human chorionic gonadotropin (hCG) treatment. The histologic samples in our study included both scrotal and inguinal testes. Using anin situ apoptosis detection method, we were able to demonstrate that both interstitial cells and germ cells were affected and that the specific germ cells undergoing apoptosis were exclusively spermatogonia. Apoptosisin situ was further seen in both scrotal and inguinal testes; in scrotal testes the numbers of apoptotic spermatogonia were 170% of those seen in the cryptorchid testes (p < 0.05). Analysis of apoptotic DNA fragmentation from isolated DNA of a few selected biopsy samples served to validate our in situ findings. The amount of germ cell apoptosis analyzed during the 1st mo after hCG treatment was increased in both scrotal and inguinal testes compared with the amount before treatment (p< 0.001). But after the 1st mo it returned to the initial level, suggesting that hCG (and/or androgen) withdrawal increases germ cell apoptosis in the human testis. Our findings lead to the conclusion that apoptosis is a hormonally controlled, normal phenomenon in a human prepubertal testis and that cryptorchidism decreases its occurrence by reducing the number of germ cells capable of undergoing apoptosis.

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.

The birth rate of hypospadias in the Turku area in Finland

Reports based on national registers of congenital malformations have suggested that the birth rate of hypospadias has increased during the last few decades. Register‐based information may, however, have pitfalls because of changes in diagnostics, reporting accuracy and registration system. The aim of this study was to determine the current birth rate of hypospadias in Turku University Central Hospital (TUCH) in Finland. This was a prospective study on live‐born boys born in TUCH from 1997 to 1999. In the total birth cohort (n=5,798) as well as in a special subcohort group (n=1,505) 0.3% of boys had hypospadias. Only one scrotal hypospadias was found in a boy who had a chromosomal anomaly. Other hypospadias were glandular or coronal. No increase was found in the birth rate of hypospadias when comparing our result with register‐based data of boys born in Finland during the years 1970 to 1986 and surgically treated for hypospadias by the age of 8 years. No difference was found either from malformation register‐based data concerning the nationwide birth rate of hypospadias during the years 1993 to 1998. Due to differences in national registration systems between countries, prospective studies with equal assessment criteria are needed in order to make reliable international comparisons.

Cryptorchidism: an indicator of testicular dysgenesis?

Cryptorchidism is a common ailment of new-born boys, affecting 1–9% of full term boys at birth. Cryptorchidism has been associated with an increased risk of testicular cancer and reduced fertility. Aetiology of cryptorchidism remains obscure in most cases. Familial occurrence suggests a heritable susceptibility to cryptorchidism; however, seasonal variation in the incidence of cryptorchidism suggests that environmental factors also contribute. Testicular descent is characterised by androgen-dependent regression of cranial suspensory ligament and androgen + insulin-like hormone 3 (Insl3)-dependent gubernacular outgrowth. Even though hormonal defects are rarely detected in patients, both hypo-and hypergonadotropic hormonal patterns have been associated with cryptorchidism. Moreover, cryptorchid boys have significantly reduced serum androgen bioactivity at 3 months of age when normal boys have a strong surge of reproductive hormones. Defects in Insl3 action cause cryptorchidism in male mice, and over-expression in female mice causes ovarian descent. Defects in leucine-rich repeat-containing G-protein-coupled receptor 8/G-protein-coupled receptor affecting testis descent (LGR8/GREAT), the receptor for Insl3, manifest the same phenotype as Insl3 knockout mutants. Even though mutations found in Insl3 and LGR8/GREAT genes are not a common cause of cryptorchidism in patients, it remains to be resolved whether low Insl3 levels during development are associated with cryptorchidism. Cryptorchidism may reflect foetal testicular dysgenesis that may later manifest as subfertility or testicular cancer.

Lack of LGR8 gene mutation in Finnish patients with a family history of cryptorchidism

Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male. Although in Western countries 1-2% of males at the age of 3 months are diagnosed with this condition, its aetiology is still unknown. Animal models suggest a possible genetic basis for this disorder. Recently, the INSL3 (Leydig insulin-like peptide) gene and its cognate receptor, LGR8, were found to be important in testicular descent by regulating gubernacular development. Male mice null for either INSL3 or LGR8 genes exhibited bilateral cryptorchidism. Because earlier studies indicated that mutation of the INSL3 gene is not associated with the development of human cryptorchidism, this study analysed whether mutations in the LGR8 gene could be associated with this disorder. Sequencing of 18 exons of the LGR8 gene in 23 cryptorchid Finnish patients and a group of 33 control subjects allowed the identification of three nucleotide changes in exons 12 and 17, showing single base substitutions from A to G at positions 957, 993, and 1810 of LGR8. Among the three changes, only the 1810 A to G substitution is associated with an amino acid change from isoleucine to valine (Ile604Val) located in the fifth transmembrane domain of this seven-transmembrane receptor. This change was more frequent in a control group of normal fertile adult males and infant boys than in the group of cryptorchid males. The change is not associated with altered receptor signalling, thus suggesting the presence of a polymorphism unrelated to the cryptorchid phenotype. These data indicate that mutations involving the human LGR8 gene do not represent a frequent cause of cryptorchidism in the Finnish population.

Natal and Neonatal Teeth in Relation to Environmental Toxicants

Infants born to mothers heavily exposed to polychlorinated biphenyls (PCBs) and dibenzofurans (PCDFs) have earlier been reported to have increased prevalences of natal and neonatal teeth. Some tendency toward higher prevalence figures of natal and neonatal teeth can be seen in the literature in normal child populations during the last 40 y. We therefore decided to determine the present prevalence of these teeth in a Finnish population and to evaluate whether infants with natal and neonatal teeth are more exposed to PCBs, PCDFs, and polychlorinated dibenzo-p-dioxins (PCDDs) than infants on average. A total of 34,457 infants born in 1997–2000 in four hospitals in southern Finland were examined for natal and neonatal teeth. The exposure of the infant to PCBs and PCDD/Fs was evaluated by measuring the levels of 17 most toxic PCDD/F and 36 PCB congeners in his or her mothers milk sample when the child was 4–8 wk old. A total of 34 infants had one or two natal (29 infants) or neonatal teeth (five infants). The milk analyses showed that the median level of PCDD/Fs as toxic equivalent (World Health Organization–recommended 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent quantity for PCDD/Fs in fat) was 11.9 pg/g in fat, and that of PCBs (World Health Organization–recommended 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent quantity for PCBs) was 7.24 pg/g in fat. These levels corresponded to the prevailing levels. The results showed that the prevalence of natal and neonatal teeth was 1:1000. No association was found between pollutant levels and occurrence of natal and neonatal teeth, indicating that the prevailing levels of PCDD/Fs and PCBs are likely to be below the threshold to cause perinatal eruption of teeth.

Inhibin gene expression in a large cell calcifying Sertoli cell tumour and serum inhibin and activin levels

Inhibin is a potential tumour suppressor gene product in the gonads. While inhibin gene products may have a role in tumourigenesis, serum inhibin levels can be used as a marker for ovarian tumours derived from granulosa cells. Tumours derived from Sertoli cells, testicular counterparts of granulosa cells, are rare. To assess whether inhibin could be used as a human Sertoli cell tumour marker, serum inhibin and activin levels and inhibin subunit mRNA expression in the testis were studied. Northern blot and in situ hybridization revealed abundant expression of inhibin α, βA, and βB subunit mRNAs in large cell calcifying Sertoli cell tumours found in a 12‐year old boy with Carney complex. The tumours were multifocal and bilateral. Serum inhibin levels were clearly elevated at the time of the diagnosis, decreased by 50% after one of the testes was removed, and were low or undetectable after the second orchidectomy six weeks later. Activin was undetectable before the orchidectomies, while a low concentration of activin‐A was measured after them. Follicle stimulating hormone (FSH) concentration increased from normal pubertal value to castration level as expected. Normal seminiferous tubules also showed inhibin subunit α and βB mRNA expression, whereas inhibin βA mRNA was expressed in normal Leydig cells. These data suggest that serum inhibin reflects Sertoli cell activity and can be used as a human tumour marker.