Fetal bisphenol A and ethinylestradiol exposure alters male rat urogenital tract morphology at birth: Confirmation of prior low-dose findings in CLARITY-BPA.

Bisphenol A (BPA) is a contaminant in virtually all Americans. To examine BPA's adverse effects, the FDA-NCTR, NIEHS, and 14 groups of academic scientists formed a consortium: CLARITY-BPA. The purpose of our study was to investigate the effects of a wide range of doses of BPA on fetal development of the NCTR CD-SD male rat urogenital sinus (UGS). Pregnant rats were administered BPA or positive control ethinylestradiol (EE2) daily, via oral gavage, from gestational day 6 through parturition. Tissues were collected on postnatal day 1 and the UGS was analyzed using computer-assisted 3-D reconstruction. Importantly, only low doses of BPA, as well as EE2, significantly changed birth weight and UGS morphology, including an increased size of the colliculus and decreased size of the urethra, consistent with prior reported BPA and EE2 effects. Our findings provide further evidence that BPA mediates nonmonotonic developmental effects on the fetal urogenital sinus.

Potential Interference of Oil Vehicles on Genital Tubercle Development during the Fetal Period in ICR Mice.

Corn oil, sesame oil, and 10% ethanol in corn oil are commonly used as dosing vehicles in toxicology studies. Since these vegetable oils contain bioactive compounds, it is important for toxicology studies to characterize the toxicities of the dosing vehicles themselves. It has been recently proposed that the width of the genital tubercle (GT), the dorsal-ventral length (D-V length) of the GT, and urethral tube closure in mouse fetuses can be used as novel markers for monitoring sexual development in mice. However, how these parameters are influenced by the dosing vehicles themselves remains unclear. Therefore, we evaluated the effects of corn oil, sesame oil, and 10% ethanol in corn oil on GT width, D-V length, and GT morphology in ICR mice. Our results showed that all three vehicles influenced GT width and D-V length, but not GT morphology, suggesting that the effects of dosing vehicles themselves might need to be considered when GT width or D-V length is used as a parameter to evaluate the effects of chemicals on GT development.

Assessing the impact of in-utero exposures: potential effects of paracetamol on male reproductive development.

Human male reproductive disorders (cryptorchidism, hypospadias, testicular cancer and low sperm counts) are common and some may be increasing in incidence worldwide. These associated disorders can arise from subnormal testosterone production during fetal life. This has resulted in a focus on in-utero environmental influences that may result in reproductive effects on the offspring in later life. Over recent years, there has been a dramatic increase in the scientific literature describing associations between in-utero environmental exposures (eg, industrial chemicals and pharmaceuticals) and subsequent reproductive outcomes in male offspring. This includes studies investigating a potential role for in-utero analgesic exposure(s) on the fetal testis; however, providing definitive evidence of such effects presents numerous challenges. In this review, we describe an approach to assessing the potential clinical relevance of in-utero (and postnatal) environmental exposures on subsequent male reproductive function using exposure to the analgesic paracetamol as an example.

Severity of birth defects after propylthiouracil exposure in early pregnancy.

BACKGROUND: Propylthiouracil (PTU) used in the treatment of maternal hyperthyroidism in early pregnancy may be associated with a higher prevalence of birth defects in the face and neck region and in the urinary system but the severity of these complications remains to be elucidated. METHODS: Review of hospital-registered cases of birth defects in the face and neck region and in the urinary system after PTU exposure in early pregnancy. We obtained information on maternal redeemed prescription of PTU and child diagnosis of birth defect from nationwide registers for all children born in Denmark between 1996 and 2008 (n=817,093). The children were followed until December 31, 2010 (median age, 8.3 years) and the Cox proportional hazards model was used to estimate adjusted hazard ratio (HR) with 95% confidence interval (CI) for having a birth defect after PTU exposure versus nonexposed children (n=811,730). RESULTS: Fourteen cases of birth defects were identified in the face and neck region and in the urinary system after PTU exposure in early pregnancy; 11 children were exposed to PTU only (n=564), whereas 3 children were born to mothers who switched from methimazole (MMI)/carbimazole (CMZ) to PTU in early pregnancy (n=159). Among children exposed to PTU only, the adjusted HR for having a birth defect in the face and neck region was 4.92 (95% CI 2.04-11.86) and in the urinary system 2.73 (1.22-6.07). Looking into details of the 14 cases, 7 children were diagnosed with a birth defect in the face and neck region (preauricular and branchial sinus/fistula/cyst) and 7 children had a birth defect in the urinary system (single cyst of kidney and hydronephrosis). Surgical treatment was registered in 6 of the cases with a birth defect in the face and neck region and 3 of the cases with a birth defect in the urinary system. Two of the children with a birth defect in the urinary system also had other birth defects (genital organs). CONCLUSIONS: We report details on possible PTU-associated birth defects. They tend to be less severe than the defects observed after MMI/CMZ exposure. Yet, the majority of affected children had to undergo surgery.

Perinatal androgenic exposure and reproductive health effects female rat offspring.

Environmental contaminants known as endocrine-disrupting chemicals (EDC) have been associated with adverse effects on reproductive processes. These chemicals may mimic or antagonize endogenous hormones, disrupting reproductive functions. Although preliminary studies focused on environmental estrogens, the presence of compounds with androgenic activity has also been described. This study examines exposure of female pregnant and lactating rats to low doses of androgens and assesses potential effects on female offspring. Pregnant Wistar rats were exposed to testosterone propionate (TP) at doses of 0.05, 0.1, or 0.2 mg/kg or corn oil (vehicle), subcutaneously, to determine influence on reproductive health of female offspring. There were two exposure groups: (1) rats treated from gestational day (GD) 12 until GD 20; and (2) animals treated from GD 12 until the end of lactation. Perinatal exposure to TP produced increased anogenital distance after birth and diminished height of uterine glandular epithelium at puberty in animals exposed to 0.2 mg/kg. However, these alterations were not sufficient to impair sexual differentiation and normal physiology of the female rat reproductive tract.

Corticosteroid-induced kidney dysmorphogenesis is associated with deregulated expression of known cystogenic molecules, as well as Indian hedgehog.

An intact genome is essential for kidney growth and differentiation, but less is known about whether, and how, an altered fetal milieu modifies these processes. Maternal low-protein diets perturb growth of the metanephros, the precursor of the mature kidney. Fetal corticosteroid overexposure may, in part, mediate this, because such diets downregulate placental 11beta-hydroxysteroid dehydrogenase-2, which degrades maternal corticosteroids. We report that glucocorticoid and mineralocorticoid receptors are expressed in mouse metanephric epithelia. Metanephroi maintained in organ culture with hydrocortisone (1.4 or 14 microM) underwent a dose-dependant deceleration of overall growth accompanied by cyst formation. Dexamethasone, a glucocorticoid, reproduced these outcomes, but aldosterone, a mineralocorticoid, did not. Hydrocortisone upregulated transcripts levels of cadherin-11 and downregulated prospero-related homeobox-1, hence mimicking reported effects of maternal low-protein diet. Hydrocortisone also upregulated transcripts encoding Na(+)-K(+)-ATPase subunits and ligands for the epidermal growth factor receptor, all previously implicated in renal cyst growth. The most upregulated transcript, however, was indian hedgehog, and the encoded protein was immunodetected in metanephric cysts. Furthermore, in the presence of hydrocortisone, cystogenesis, but not whole organ growth, was significantly reduced by cyclopamine, a drug downregulating hedgehog signaling. Finally, both glucocorticoid receptor and indian hedgehog proteins were detected by immunohistochemistry in cystic tubules within human dysplastic kidneys, consistent with the hypothesis that these molecules modify the severity of this congenital malformation. Collectively, our observations raise the possibility that enhanced hedgehog signaling is an important stimulus for renal cyst formation. Furthermore, pharmacological inhibition of this pathway should be explored as a potential therapy for renal cystic diseases, starting with relevant animal models.

Urogenital abnormalities in men exposed to diethylstilbestrol in utero: a cohort study.

BACKGROUND: Diethylstilbestrol (DES), a synthetic estrogen widely prescribed to pregnant women during the 1940s70s, has been shown to cause reproductive problems in the daughters. Studies of prenatally-exposed males have yielded conflicting results. METHODS: In data from a collaborative follow-up of three U.S. cohorts of DES-exposed sons, we examined the relation of prenatal DES exposure to occurrence of male urogenital abnormalities. Exposure status was determined through review of prenatal records. Mailed questionnaires (1994, 1997, 2001) asked about specified abnormalities of the urogenital tract. Risk ratios (RR) were estimated by Cox regression with constant time at risk and control for year of birth. RESULTS: Prenatal DES exposure was not associated with varicocele, structural abnormalities of the penis, urethral stenosis, benign prostatic hypertrophy, or inflammation/infection of the prostate, urethra, or epididymus. However, RRs were 1.9 (95% confidence interval 1.13.4) for cryptorchidism, 2.5 (1.54.3) for epididymal cyst, and 2.4 (1.54.4) for testicular inflammation/infection. Stronger associations were observed for DES exposure that began before the 11th week of pregnancy: RRs were 2.9 (1.65.2) for cryptorchidism, 3.5 (2.06.0) for epididymal cyst, and 3.0 (1.75.4) for inflammation/infection of testes. CONCLUSION: These results indicate that prenatal exposure to DES increases risk of male urogenital abnormalities and that the association is strongest for exposure that occurs early in gestation. The findings support the hypothesis that endocrine disrupting chemicals may be a cause of the increased prevalence of cryptorchidism that has been seen in recent years.

Estrogen-induced uterine abnormalities in TIMP-1 deficient mice are associated with elevated plasmin activity and reduced expression of the novel uterine plasmin protease inhibitor serpinb7.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein capable of regulating a variety of biological processes in a wide array of tissue and cell types. We have previously demonstrated that TIMP-1 deficient mice exhibit alterations in normal uterine morphology and physiology. Most notably, absence of TIMP-1 is associated with an altered uterine phenotype characterized by profound branching of the uterine lumen and altered adenogenesis. To begin to assess the mechanism by which TIMP-1 may control these uterine events, we utilized steroid-treated ovariectomized wild-type and TIMP-1 null mice exposed to estrogen for 72 hr. Administration of estrogen to TIMP-1 deficient mice resulted in development of an abnormal uterine histo-architecture characterized by increased endometrial gland density, luminal epithelial cell height, and abnormal lumen structure. To determine the mediators which may contribute to the abnormal uterine morphology in the TIMP-1 deficient mice, cDNA microarray analysis was performed. Analysis revealed that expression of two plasmin inhibitors (serpbinb2 and serbinb7) was significantly reduced in the TIMP-1 null mice. Associated with the reduction in expression of these inhibitors was a significant increase in plasmin activity. Localization of the novel uterine serpinb7 revealed that expression was confined to the luminal and glandular epithelial cells. Further, expression of uterine serpinb7 was decreased by estrogen and showed an inverse relationship with plasmin activity. We conclude from these studies that in addition to controlling MMP activity, TIMP-1 may also control activity of serine proteases through modulation of serine protease inhibitors such as serpinb7.

[A morphological study with serial histological slices on the normal and abnormal gubernacula in newborn male mice].

OBJECTIVE: To explore the feasibility of serial slices microscopic histological investigation for the elaborate evaluation of reproductive system malformations. METHODS: Newborn male mice prenatally exposed to different doses of subcutaneously given diethylstilbestrol (DES) from gestational day 9 to 17 were treated by fixing parts of the abdomen in situ and setting them to transected serial slices. All the slices were stained, studied under the microscope and serially recorded by software. The gubernaculum was morphologically analyzed and its location and size were measured. RESULTS: Morphologically, the gubernaculum could be identified clearly, its structure inhomogeneous from proximal to distal and dissymmetric from right to left. The environmental estrogen produced different effects on the morphology of the gubernaculum in different parts and most obviously affected its length. CONCLUSION: Prenatal exposure to environmental estrogen has evident and general effects on the gubernacular development of newborn male mice. The morphological study with serial histological slices gives a precise and systematic evaluation of genital malformations.

Impaired reproductive development in sons of women occupationally exposed to pesticides during pregnancy.

OBJECTIVES: The aim of this prospective study was to investigate whether occupational pesticide exposure during pregnancy causes adverse effects on the reproductive development in the male infants. DESIGN AND MEASUREMENTS: Pregnant women employed in greenhouses in Denmark were consecutively recruited, and 113 mother-son pairs were included. The mothers were categorized as occupationally exposed (91 sons) or unexposed (22 sons) to pesticides during pregnancy. Testicular position and volume, penile length, and position of urethral opening were determined at 3 months of age using standardized techniques. Concentrations of reproductive hormones in serum from the boys were analyzed. RESULTS: The prevalence of cryptorchidism at 3 months of age was 6.2% [95% confidence interval (CI), 3.0-12.4]. This prevalence was considerably higher than among Danish boys born in the Copenhagen area (1.9%; 95% CI, 1.2-3.0) examined by the same procedure. Boys of pesticide-exposed mothers showed decreased penile length, testicular volume, serum concentrations of testosterone, and inhibin B. Serum concentrations of sex hormone-binding globulin, follicle-stimulating hormone, and the luteinizing hormone:testosterone ratio were increased compared with boys of nonexposed mothers. For individual parameters, only the decreased penile length was statistically significant (p = 0.04). However, all observed effects were in the anticipated direction, and a joint multivariate test showed that this finding had a p-value of 0.012. CONCLUSIONS: Our findings suggest an adverse effect of maternal occupational pesticide exposure on reproductive development in the sons despite current greenhouse safeguards and special measures to protect pregnant women.

In utero exposure to the antiandrogen 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) in relation to anogenital distance in male newborns from Chiapas, México.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is still used for disease control in some areas, resulting in high levels of human exposure. The main degradation product of DDT is 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), an antiandrogen. In animal experiments, in utero exposure to DDE decreases anogenital distance in male offspring. In these models, anogenital distance serves as a measure of fetal androgen action. The authors designed the present study to examine the hypothesis that in utero exposure to DDE decreases anogenital distance in newborn human males. A cross-sectional study of 781 newly delivered male infants was conducted in 2002-2003 in Chiapas, México, where DDT had recently been used for malaria control. Measurements of anogenital distance and penile dimensions were taken, and a sample of the mother's blood was drawn. In this population, the range of serum DDE levels was large (0.8-398 microg/liter). The authors, using two-sided tests, found no evidence that exposure in utero to DDE was related to reduced androgen action as reflected by anogenital distance or penile dimensions at birth. If DDE has important antiandrogenic action in humans, it may be manifest only at higher levels of exposure or via effects on other outcomes.

Pregnant "DES daughters" and their offspring.

QUESTION: I am a 34-year-old woman in my second trimester of pregnancy. My mother took diethylstilbestrol when she was pregnant with me. Could my expected child be affected by this? ANSWER: Animal studies suggest the child could be affected, but little data on humans strongly support this. You could plan to have your child monitored for a potential, though unlikely, effect.

Esophageal atresia and other visceral anomalies in a modified Adriamycin rat model and their correlations with amniotic fluid volume variations.

The Adriamycin rat model (ARM) has been used to produce visceral malformations in fetuses to explain the mechanisms of foregut division. The models vary in the dosage of Adriamycin (ADR) and in the number of applications. Our study of a modified ARM using 2.2 mg/kg of ADR for 2 days only, intraperitoneally in pregnant rats, is presented. A total of 81 fetuses were obtained with this model from the ADR group, 74 (91%) alive. Uretero-hydronephrosis (UHN) was observed in 70 fetuses (95%), esophageal atresia (EA) in 68 (92%), duodenal atresia (DA) in 68 (92%), bladder hypoplasia (BH) in 67 (90%), plus other malformations. In evaluating amniotic fluid (AF) volume of the fetuses with EA with tracheo-esophageal fistula (TEF) (group I) and EA without TEF (group II), both associated with bilateral UHN when compared with the control group (group III), groups I and II showed higher AF volume in groups I and II than the control group (group III) did ( p=0.0001). In conclusion, ARM was adequate to produce EA and other visceral malformations. The use of ADR in a higher dosage for a shorter period of time produced better results than those presented in previous literature. The increase of AF volume obtained in fetuses presenting EA plus bilateral UHN strongly suggests, despite ureteral dilatation (urinary obstruction), that a malformed communication may exist between the urinary system and the amniotic cavity, permitting the existence of polyhydramnios that is due to digestive obstruction such as EA and DA.

Perturbed medullary tubulogenesis in neonatal rat exposed to renin-angiotensin system inhibition.

BACKGROUND: Pharmacological interruption of the angiotensin II type-1 receptor (AT(1)) signalling during nephrogenesis in rats induces irreversible abnormalities in kidney morphology, comprising papillary atrophy and tubulointerstitial damage, which are characterized by tubular dilatation/atrophy and interstitial inflammation/fibrosis. This study determined the time course for development of tubular structural and inflammatory changes and possible cytokine production in the renal medulla of newborn rats exposed to angiotensin-converting enzyme (ACE) inhibition. Additionally, medullary expression of E-cadherin, a marker for tubular formation, was investigated in ACE-inhibited rats. METHODS: Newborn rats were exposed (postnatal days 0-12) to ACE inhibitor enalapril and killed at days 1, 2, 4, 9 and 13. One kidney was used for morphological evaluation and the other for immunohistochemistry, using antibodies directed against monocytes/macrophages, T cells and E-cadherin on frozen sections. In a separate experiment, rats were treated for 9 days and had their kidneys processed for western immunoblot and immunohistochemistry, where antibodies directed against monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha) were used on paraffin sections. RESULTS: In renal medulla from enalapril-treated rats, volume fractions of tubular lumens and interstitium were increased from postnatal days 2 and 4, respectively, while that of tubular cells was decreased from 4 days of age. Concomitant loss and/or reduction in E-cadherin expression (from day 2) was observed in dilated medullary tubules of enalapril-treated rats. Furthermore, in the medulla of enalapril-treated rats, the increased number of ED2+ (resident macrophages) cells, followed by the increase in ED1+ (monocytes/macrophages) and CD4+ T cells, was observed at days 9 and 13, respectively. This was accompanied by increased medullary expression of TNF-alpha at day 9. CONCLUSIONS: Neonatal ACE inhibition perturbs medullary tubulogenesis, as indicated by tubular dilatation and a lack of E-cadherin expression in these tubules. Macrophage/monocyte-mediated immune response is a secondary event, coincidentally associated with the up-regulation of TNF-alpha.

Relationship of the fistulas to the rectum and genitourinary tract in mouse fetuses with high anorectal malformations induced by all-trans retinoic acid.

Since high anorectal malformations with fistulae in human embryos and fetuses of successive developmental stages have not been reported, the embryologic relationship between the rectal fistula (RF) and the genitourinary tract (GUT) in high anorectal agenesis (ARA) remains to be elucidated. This study investigates the developmental relationship between the RF and the GUT in male and female fetuses with high ARA using our established model for high ARA with fistula in mice. Pregnant mice received all-trans retinoic acid suspended in corn oil (5 mg/ml) 100 mg/kg i.p. on day 9 of pregnancy. All fetuses were removed from the uterus on a single day from days 12 to 18 of pregnancy. The caudal regions were analyzed histologically with hematoxylin and eosin staining. All fetuses examined had high ARA with fistula. On day 12 of pregnancy, an anomalous communication was seen between the urogenital sinus (UGS) and the rectum. In the affected female fetuses, on day 14 of pregnancy the paramesonephric (müllerian) ducts and müllerian tubercle were located above the rectocloacal fistula (RCF), and on day 18 of pregnancy the uterovaginal canal was located between the cloaca and the RCF. In the male fetuses, on day 14 of pregnancy the junction between the mesonephric (wolffian) duct and the UGS was located away from the junction between the rectum and the UGS. On day 18 of pregnancy the ejaculatory duct was located between the urinary bladder and the rectourethral fistula. The results of our experiment clearly show the embryologic relationship between the RF and the GUT with high ARA. The anomalous communication between the UGS and the rectum may interfere with normal caudal migration along the dorsal wall of the UGS at the junction between the UGS and the mesonephric or paramesonephric duct.

Cloacal and urogenital malformations in adriamycin-exposed rat fetuses.

OBJECTIVES: To determine the spectrum of cloacal and urogenital malformations in rat fetuses prenatally exposed to adriamycin, which causes abnormalities that strongly resemble the VATER association (vertebral defects, anal atresia, tracheo-oesophageal fistula with oesophageal atresia, renal defects and radial limb dysplasia) in humans, and to evaluate how closely such anomalies resemble those seen in humans. MATERIALS AND METHODS: Timed-pregnant rats were injected intraperitoneally with adriamycin at 6-9 days of gestation; a control group received saline only. Fetuses (35 treated and 30 control) were recovered at 21 days of gestation, and examined macroscopically and microscopically for cloacal and urogenital abnormalities. RESULTS: All the treated fetuses had no bladders and severe hydroureter/hydronephrosis on one or both sides. Male fetuses had a proximal blind-ending urethra communicating with dilated ureters and giving rise to vasa. Female fetuses had a persistent urogenital sinus communicating with the ureters and cervix/uterus; 57% of the treated group had an imperforate anus and some had recto-urethral fistulae (males) or recto-urogenital fistulae (females). CONCLUSION: Rat fetuses exposed to adriamycin have a spectrum of cloacal and urogenital anomalies (predominantly no bladder) which is more severe than those of the human VATER association. Nonetheless, this is an excellent animal model for the study of genitourinary embryology, especially bladder development.

Critical timing of bladder embryogenesis in an adriamycin-exposed rat fetal model: a clue to the origin of the bladder.

BACKGROUND/PURPOSE: Administration of Adriamycin (ADR) in utero to pregnant rats (vaginal plug, day 0) on gestational days (GD) 6 to 9 resulted in the offspring having a cluster of malformations, including absence of bladder in 100% of cases. This study aimed to determine the critical timing of the embryological window in bladder development in this animal model. METHODS: Timed-pregnant rats were divided randomly and injected intraperitoneally with ADR at 2 mg/kg on GD 6 to 9; GD 7 to 10; GD 8 to 11; GD 9 to 12; GD 6,8, and 9 (missing GD 7); and GD 6, 7, and 9 (missing GD 8). The control group received saline. Fetuses were harvested near term on GD 21 and dissected under a dissecting microscope and examined for gross anorectal and urogenital anomalies. RESULTS: Administration of ADR on GD 6 to 9 (n = 63); GD 7 to 10 (n = 42); and GD 6, 7, and 9 (n = 35) resulted in 100%, 83%, and 77% bladder agenesis respectively, in contrast with 53% and 26% on GD 8 to 11 (n = 36) and GD 6, 8, and 9 (n = 49), respectively. The control (n = 52) and the GD 9 to 12 (n = 27) groups all had normal bladder development. The proportion of other urogenital and anorectal anomalies mirror that of bladder agenesis. CONCLUSION: The results showed GD 7 to be the critical embryological timing in which bladder development can be affected by ADR, possibly by targeting the gene that is expressed in the embryonic bladder during this narrow time interval.