Reduction In Anogenital Distance Research Articles

Effects of low-dose bisphenol AF on mammal testis development via complex mechanisms: alterations are detectable in both infancy and adulthood.

Despite growing concern about adverse effects of bisphenol AF (BPAF) due to its endocrine disrupting properties, there is a lack of toxicity data from low-dose studies and direct evidence linking its adverse effects to endocrine disrupting properties. Here, we investigated the effects of gestational and postnatal exposure to BPAF through drinking water (0.15-15μg/mL, equivalent to the daily intake of ~ 50 and 5mg/kg/day) on testis development in mice. We found that like mestranol, 5mg/kg/day BPAF resulted in remarkable decreases in multiple male reproductive parameters in adulthood, such as the sperm number and serum testosterone level. Notably, 50μg/kg/day BPAF also caused significant decreases in anogenital distance (AGD), the luteinizing hormone level and spermatocyte number, along with declining trends in sperm number and the serum levels of testosterone and follicle-stimulating hormone. In line with the adverse outcomes observed in adulthood, on postnatal day (PND) 9, we also observed BPAF-caused dose-dependent alterations, including reduced AGD, seminiferous tubule area and numbers of total germ cells, spermatocytes and Leydig cells, coupled with down-regulated expression of male-biased genes in testes. Even when exposure to 5mg/kg/day BPAF as well as MES was initiated from PND 0, similar alterations in male reproductive parameters were also found on PND 9, along with a decrease in the GnRH content in the hypothalamus; moreover, testicular alterations and the reduction in AGD were partly antagonized by the estrogen receptor (ER) antagonist ICI 182,780, but the reduction of GnRH production was not done, showing that the effects of BPAF on testis development may be partially mediated by ER signaling. In conclusion, all the findings demonstrate that low-dose BPAF can partly disrupt mammal testis development and cause adverse testicular outcomes in adulthood, indicating a potential reproductive risk to mammals including humans. Importantly, our finding that developmental alterations elicited by BPAF have been detectable on PND 9 provides important motivation for the development of effective methods for early detection of adverse effects of estrogenic chemicals on testis development.

Using deep learning to track 3D kinematics

In recent years methamphetamine (MA) use has become more prevalent, and of particular concern is its growing popularity of MA among women of childbearing age. However, to date, studies examining MA effects on the developing offspring in laboratory animals are limited. Thus, the aim of this study was to determine if in utero MA exposure in rats at pharmacological doses can have a negative impact on neonatal neurodevelopment and behaviour. Pregnant Sprague-Dawley dams (n = 10 dams/group) received MA (0, 0.625, 1.25, 2.5 mg/kg) once daily via oral gavage from gestational day 7 to 21. Maternal body weight, food and water consumption were recorded daily. A range of standard neurodevelopment parameters was examined in the offspring during the neonatal period. There were no neurodevelopmental deficits observed with offspring exposed to 0.625 mg/kg MA, in fact, there were enhancements of neurodevelopment in some parameters at this low dose. However, exposure to the 1.25 mg/kg MA dose resulted in significant impairments in surface righting reflex and forelimb grip in both sexes. Exposure to the 2.5 mg/kg MA dose resulted in a significant reduction in ano-genital distance in males, and in both sexes resulted in delayed fur appearance and eye opening, impairments in surface righting reflex and negative geotaxis, and a reduction in body length. In conclusion, this study demonstrates that pharmacologically relevant doses of MA can have profound dose-related effects on neonatal outcome. If extrapolated to the clinical scenario this will give cause for concern regarding the risks associated with this drug of abuse at relatively low doses.

In utero exposure to persistent and nonpersistent endocrine-disrupting chemicals and anogenital distance. A systematic review of epidemiological studies†.

Anti-androgenic endocrine-disrupting chemicals (EDCs) can cross the placenta to modify early offspring sexual dimorphic markers. These changes are linked to anogenital distance (AGD), which is an androgen-sensitive anthropometric parameter used as a biomarker of perineal growth and caudal migration of the genital tubercle. This review aimed to summarize strength of evidence for associations of in utero exposure to EDCs with AGD and to identify gaps and limitations in the literature so as to inform future research. We performed an electronic search of English literature in September 2019 in medical literature analysis and retrieval system online (MEDLINE), Web of Science and Toxline. We included epidemiological studies that examined in utero exposure to persistent and nonpersistent EDCs and considered AGD in offspring as an outcome. Our review contained 16 investigations examining exposure to persistent EDCs (nine studies) and nonpersistent EDCs (seven studies). Some individual studies reported an inverse association between exposure to bisphenol A (BPA), dioxins, perfluoroalkyl substances, and organochlorides and AGD in both male and female offspring. Meta-analysis of three studies found a small reduction of AGD in female offspring exposed to BPA. The number of studies per chemical is small, and number of subjects examined is limited; so, replication of these results is needed. To achieve more specificity and better replication of results, future studies should establish the association of nonpersistent EDCs using multiple urine samples, evaluate the cumulative impact of exposure to a mixture of anti-androgenic chemicals, and offer adequate consideration of more maternal- and children-related confounding factors.

Impact of intrauterine exposure to betamethasone on the testes and epididymides of prepubertal rats

Therapy with betamethasone, a synthetic glucocorticoid, is used in cases of preterm birth risk, in order to promote fetal lung maturation, and decrease neonatal mortality and morbidity. However, late reproductive disorders related to the prenatal exposure to this compound have been reported by our Laboratory, in both male and female rats. Thus, the present study aimed to evaluate the impact of betamethasone on postnatal reproductive development, during pre-puberty, of male offspring exposed in utero to this synthetic glucocorticoid. For this purpose, pregnant Wistar rats were allocated into two groups: Control, treated with saline, and the group treated with betamethasone at 0.1 mg/kg/day. Control and betamethasone groups were treated with intramuscular injection on gestational days 12, 13, 18 and 19, critical days of prenatal reproductive development. The treatment is associated with reduced body and organ weights, disorders in initial reproductive parameters of pre-pubertal male offspring exposed in utero to betamethasone, such as reduction of anogenital distance, alterations in histomorphometric parameters and immunostaining pattern of androgen and estrogen receptors on testicles and epididymides. Our results suggest that prenatal exposure to betamethasone potentially causes reproductive reprogramming and impairs male postnatal reproductive development. This data raise concerns about the use of betamethasone for human antenatal therapy.

Experimentally induced testicular dysgenesis syndrome originates in the masculinization programming window.

The testicular dysgenesis syndrome (TDS) hypothesis, which proposes that common reproductive disorders of newborn and adult human males may have a common fetal origin, is largely untested. We tested this hypothesis using a rat model involving gestational exposure to dibutyl phthalate (DBP), which suppresses testosterone production by the fetal testis. We evaluated if induction of TDS via testosterone suppression is restricted to the "masculinization programming window" (MPW), as indicated by reduction in anogenital distance (AGD). We show that DBP suppresses fetal testosterone equally during and after the MPW, but only DBP exposure in the MPW causes reduced AGD, focal testicular dysgenesis, and TDS disorders (cryptorchidism, hypospadias, reduced adult testis size, and compensated adult Leydig cell failure). Focal testicular dysgenesis, reduced size of adult male reproductive organs, and TDS disorders and their severity were all strongly associated with reduced AGD. We related our findings to human TDS cases by demonstrating similar focal dysgenetic changes in testes of men with preinvasive germ cell neoplasia (GCNIS) and in testes of DBP-MPW animals. If our results are translatable to humans, they suggest that identification of potential causes of human TDS disorders should focus on exposures during a human MPW equivalent, especially if negatively associated with offspring AGD.

Impairment of microvascular angiogenesis is associated with delay in prostatic development in rat offspring of maternal protein malnutrition

Experimental data demonstrated the negative impact of maternal protein malnutrition (MPM) on rat prostate development, but the mechanism behind the impairment of prostate growth has not been well understood. Male Sprague Dawley rats, borned to dams fed a normal protein diet (CTR group, 17% protein diet), were compared with those borned from dams fed a low protein diet (6% protein diet) during gestation (GLP group) or gestation and lactation (GLLP). The ventral prostate lobes (VP) were removed at post-natal day (PND) 10 and 21, and analyzed via different methods. The main findings were low birth weight, a reduction in ano-genital distance (AGD, a testosterone-dependent parameter), and an impairment of prostate development. A delay in prostate morphogenesis was associated with a reduced testosterone levels and angiogenic process through downregulation of aquaporin-1 (AQP-1), insulin/IGF-1 axis and VEGF signaling pathway. Depletion of the microvascular network, which occurs in parallel to the impairment of proliferation and differentiation of the epithelial cells, affects the bidirectional flux between blood vessels impacting prostatic development. In conclusion, our data support the hypothesis that a reduction in microvascular angiogenesis, especially in the subepithelial compartment, is associated to the impairment of prostate morphogenesis in the offspring of MPM dams.

Maternal use of mild analgesics during pregnancy associated with reduced anogenital distance in sons: a cohort study of 1027 mother-child pairs.

Is maternal use of mild analgesics in pregnancy associated with anogenital distance (AGD)-the distance from the anus to the genitals-in the offspring? Maternal use of mild analgesics [especially simultaneous use of paracetamol and nonsteroidal anti-inflammatory drugs (NSAIDs)] during pregnancy was associated with a shorter AGD in boys whereas no effect was found in girls. Mild analgesics including paracetamol (acetaminophen) and NSAIDs (e.g. ibuprofen and acetyl salicylic acid) have endocrine disrupting properties and in utero exposure reduces AGD in male rats. In humans, maternal exposure has been associated with cryptorchidism and hypospadias in male offspring but no studies have examined AGD. A prospective birth cohort study. Between 2010 and 2012, 2500 pregnant women were recruited from the Odense Child Cohort. Children were examined 3 months after the expected date of birth. Pregnant women were asked about use of medication including mild analgesics (paracetamol and NSAID) during pregnancy at recruitment (gestational age (GA) week 10-27) and at GA week 28. AGD and penile width were measured 3 months after expected date of birth by trained personnel. A total of 1027 women answered both questionnaires and their children were examined. Associations between prenatal exposure to mild analgesics and AGD and penile width were estimated using multivariable linear regression adjusting for age and weight-for-age SD score. A total of 40% of the women reported use of paracetamol and/or NSAIDs (4.4%) during the first 28 weeks of pregnancy. Exposure to analgesics during pregnancy was associated with a reduced AGD in boys, although statistically significant only for NSAIDs. The association was significant among 20 boys exposed to both paracetamol and NSAIDs (AGD -4.1 mm; CI 95%: -6.4; -1.7). Maternal intake of analgesics did not show any clear association with AGD in female offspring. No effect on penile width was found. Only 27 boys and 18 girls were exposed to NSAIDs and most of them were also exposed to paracetamol. This makes it impossible to distinguish between exposures to NSAIDs alone and a potential mixture effect. Moreover, use of mild analgesics was self-reported up to 2 months after intake, which could have caused misclassification of exposure but is probably not associated with AGD as this was unknown to the women at time of reply to the questionnaire thereby underestimating the association. Confounding by indication may also explain our findings, as the condition for which the analgesic was taken may be associated with a reduction in AGD, rather than the use of the analgesic medication. This is the first study to report such an association in humans and further studies are needed to confirm our findings. A negative association was observed between exposure to analgesics during pregnancy and AGD in boys, suggesting disruption of androgen action. The health implications of a shorter AGD are still uncertain, but in cross-sectional studies among adult men a shorter AGD is associated with poorer semen quality and lower testosterone. As 41% of the women used these painkillers the finding are of public health importance and pregnant women should be advised about the potentially harmful effects of painkiller use. The study was funded by the Danish Environmental Protection Agency by way of the Center on Endocrine Disruptors Danish Center for Hormone Disrupting Chemicals, the Danish Foundation for Scientific Innovation and Technology (09-067180), the Danish Research Council (4004-00352B_FSS), Novo Nordic Foundation (NNF15OC0017734), Ronald McDonald Children Foundation, K.A. Rohde's and wife's Foundation, Odense University Hospital and Region of Southern Denmark, Municipality of Odense, the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (2101-08-0058), Odense University Hospital Research Foundation and Odense Patient data Exploratory Network (OPEN). The authors declare they have no competing interests. Not applicable.

Prenatal exposure to perfluoroalkyl substances and anogenital distance at 3 months of age in a Danish mother-child cohort

In the Odense child cohort, serum concentrations of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) were measured in 638 pregnant women. Birth weight, head and abdominal circumferences and gestational age were determined. Anogenital distance (AGD), the distance from the anus to the genital organs, and penile width were measured 3 months after expected date of birth in 511 children. PFOS, PFHxS, PFNA and PFDA were associated with a decreased AGD in girls (p-trend<0.05) after adjusting for age and weight-for-age standard deviation score. PFOS in the highest quartile was associated with a 2.8mm (95% confidence intervals −4.5; −1.1) reduction in AGD in girls. No such tendencies were seen in boys. However, a tendency toward increased birth weight in girls and reduced birth weight in boys suggests that sex-dimorphic effects may occur from endocrine disrupting effects of these substances.

Breastfeeding Duration and Anogenital Distance in 2-Year-Old Infants.

The anogenital distance (AGD) is an anthropometric marker determined by exposures to androgens in utero and throughout the first few months of life. Early exposures to endocrine-disrupting chemicals such as phthalates have been significantly associated with shortened AGD in boys. Limited studies have explored phthalate concentrations in breast milk and infant formula. To explore the associations between breastfeeding duration and AGD measures in infants. MALAMA (Medio Ambiente y Lactancia Materna) is a follow-up study of 430 mother-child pairs, from birth to 2 years, from two population-based cohorts in Murcia, Spain. Data were collected through medical visits and telephone surveys from birth to 2 years of age. World Health Organization breastfeeding definitions were used. AGD measurements were assessed in a subsample of 71 boys and 49 girls at the 2-year visit. Descriptive analyses, Pearson correlations, and linear regressions were calculated between AGD and breastfeeding duration. Duration of all types of breastfeeding, especially full breastfeeding (FB), is correlated with AGD measures in boys (p < 0.05). AGDAS (anoscrotal distance) and AGDAP (anopenile distance) were positively associated with FB (β = 0.004, 95%CI: 0.001-0.007 and β = 0.003, 95%CI: 0.000-0.007, respectively). A positive correlation between AGD in male infants and the duration of breastfeeding is reported. Inversely, early introduction of infant formula could lead to the reduction of AGD in boys.

WITHDRAWN: Prenatal exposure to perfluoroalkyl substances and anogenital distance at 3 months of age as marker of endocrine disruption

In the Odense child cohort, serum concentrations of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) were measured in 649 pregnant women at approximately 12 weeks of gestation. Birth weight, head and abdominal circumferences were measured, and gestational age determined. Anogenital distance (AGD), i.e., the distance from the anus to the genital organs, penile width and body weight were measured 3 months after the expected date of birth in 511 children. PFOS, PFHxS, PFNA and PFDA were associated with a decreased AGD in girls (p-trend<0.05) after adjusting for age and weight-for-age standard deviation score. Specifically, PFOS in the highest quartile was associated with a 2.8mm (95% confidence intervals -4.5;-1.1) reduction in AGD in girls. No such tendencies were seen in boys. However, a tendency toward increased birth weight in girls and reduced in boys suggests that sex-dimorphic effects may occur from endocrine disrupting effects of these substances.

Shorter anogenital distance correlates with the severity of hypospadias in pre-pubertal boys.

Do pre-pubertal boys with hypospadias have a shorter anogenital distance (AGD) than boys with normal genitalia? AGD is significantly shorter in boys with hypospadias and decreases with the severity of hypospadias. Animal studies have shown that androgen disruption and exposure to endocrine disrupting chemicals during a critical time period in early gestation, termed the male programming window (MPW), result in hypospadias and reduced AGD; and the severity of hypospadias correlates with the reduction in AGD. However, this correlation has not been established in humans. A prospective descriptive study involving measurement of AGD in pre-pubertal boys (n = 458) presenting to our pediatric urology clinic with hypospadias and normal genitalia was performed over a period of 3 years. AGD was measured in pre-pubertal boys from 5 months to 14 years of age presenting to our clinic with hypospadias (n = 180: four were excluded) and compared with randomly selected boys with normal genitalia (controls, n = 274). Three variants of AGD, from the midpoint of the anus to base of the scrotum (AGD-AS), to the anterior base of penis (AGD-1) and to the posterior base of penis (AGD-2), were measured and assessed for correlation with the severity of hypospadias. Severity of hypospadias was classified as anterior, middle and posterior according to the meatal location. No significant difference in weight (P = 0.123), age (P = 0.162) or height (P = 0.591) between the two groups was observed. Only AGD-AS was significantly shorter in boys with hypospadias compared with controls (mean ± SD: 40.6 ± 9.7 mm versus 45.6 ± 9.4 mm, P < 0.001). This relation persisted after adjusting AGD for weight, height and age (β = 0.016, 95% confidence interval: 0.10-0.21; P < 0.001). The Spearman test showed a significant negative correlation for the severity of hypospadias with all the three AGD measures. Analysis of variance between anterior, middle and posterior subgroups showed a significant reduction in mean AGD-AS (P = 0.003) and AGD-2 (P = 0.008). No data were collected pertaining to in utero exposure to endocrine disrupting chemicals (EDCs) or cigarette smoke, or current diet and environmental exposure to EDCs, which may have influenced the AGD. Family history of genital malformation and use of IVF were not known. There may have been a selection bias as only boys presenting to our clinic were included. The findings suggest that prenatal androgens during early gestation influence development of the male reproductive system and support the existence of a MPW in humans. Of the three AGDs, AGD-AS may be the most reliable biomarker of this in utero androgen action. However, no direct link to any specific exposure leading to shortened AGD in pre-pubertal boys with hypospadias could be determined. Further large scale multi-center studies are needed to understand this association better. Funding was from the Hypospadias Foundation. No conflicts of interest to disclose.

Anogenital distance plasticity in adulthood: implications for its use as a biomarker of fetal androgen action.

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed.

Methamphetamine exposure during pregnancy at pharmacological doses produces neurodevelopmental and behavioural effects in rat offspring

In recent years methamphetamine (MA) use has become more prevalent, and of particular concern is its growing popularity of MA among women of childbearing age. However, to date, studies examining MA effects on the developing offspring in laboratory animals are limited. Thus, the aim of this study was to determine if in utero MA exposure in rats at pharmacological doses can have a negative impact on neonatal neurodevelopment and behaviour. Pregnant Sprague-Dawley dams (n=10 dams/group) received MA (0, 0.625, 1.25, 2.5mg/kg) once daily via oral gavage from gestational day 7 to 21. Maternal body weight, food and water consumption were recorded daily. A range of standard neurodevelopment parameters was examined in the offspring during the neonatal period. There were no neurodevelopmental deficits observed with offspring exposed to 0.625mg/kg MA, in fact, there were enhancements of neurodevelopment in some parameters at this low dose. However, exposure to the 1.25mg/kg MA dose resulted in significant impairments in surface righting reflex and forelimb grip in both sexes. Exposure to the 2.5mg/kg MA dose resulted in a significant reduction in ano-genital distance in males, and in both sexes resulted in delayed fur appearance and eye opening, impairments in surface righting reflex and negative geotaxis, and a reduction in body length. In conclusion, this study demonstrates that pharmacologically relevant doses of MA can have profound dose-related effects on neonatal outcome. If extrapolated to the clinical scenario this will give cause for concern regarding the risks associated with this drug of abuse at relatively low doses.

Effects of In Utero Exposure to D-004, a Lipid Extract from Roystonea regia Fruits, in the Male Rat: A Comparison with Finasteride

D-004 is a lipid extract obtained from Cuban royal palm fruits, consisting of a mixture of free fatty acids, that prevents prostate hyperplasia induced with testosterone in rodents. This study investigated the possible alterations due to D-004 of androgen-dependent development after exposure in utero and compared them with those due to finasteride. Rats were randomized into five experimental groups: a control group, three groups treated with D-004 at 500, 750, or 1,000 mg/kg/day, respectively, and a group treated with finasteride (10 mg/kg/day). Male rats were treated 10 weeks before and during mating. Female rats were treated for 15 days prior mating, during mating, during pregnancy, and until lactation (day 21) except for those treated with finasteride, which were only administered the drug on gestational days 12-21. All male offspring were monitored individually until necropsy after postnatal day 90. The results of the present study indicate that D-004 induced no alterations in androgen-dependent development after the exposure in utero. Also, the current study demonstrated a permanent reduction in anogenital distance and retention of nipples in adult male rats exposed to finasteride during late gestation. Significant alterations induced by exposure to finasteride were mainly in tissues dependent on dihydrotestosterone during development.

Comparative Contribution of the Aryl Hydrocarbon Receptor Gene to Perinatal Stage Development and Dioxin-Induced Toxicity Between the Urogenital Complex and Testis in the Mouse1

TCDD (2,3,7,8-tetrachlorodebenzo-p-dioxin) requires the presence of the aryl hydrocarbon receptor (Ahr) gene for its toxic effects, such as reproductive disorders in male offspring of maternally exposed rats and mice. To study the involvement of the Ahr gene in producing the toxic phenotype with respect to testicular development, we administered a relatively high dose of TCDD to mice with three different maternally derived Ahr genotypic traits, and then compared several Ahr-dependent alterations among male reproductive systems on Postnatal Day 14. Reduction in anogenital distance and expression of prostatic epithelial genes in the urogenital complex (UGC) were detected in Ahr(+/+) and Ahr(+/-) mice exposed to TCDD, whereas no difference was observed in Ahr(-/-) mice. In situ hybridization revealed the absence of probasin mRNA expression in the prostate epithelium, despite the obvious development of prostatic lobes in TCDD-exposed mice. In contrast to obvious prostatic dysfunction and induction of cytochrome P450 (CYP) family genes in the UGC by TCDD, no alterations in testicular functions were observed in germ cell/Sertoli cell/interstitial cell marker gene expression or CYP family induction. No histopathological changes were observed among the three genotypes and between control and TCDD-exposed mice. Therefore, mouse external genitalia and prostatic development are much more sensitive to TCDD treatment than testis. Further, the Ahr gene, analyzed in this study, does not significantly contribute to testicular function during perinatal and immature stages, and the developing mouse testis appears to be quite resistant to TCDD exposure.

Speaker abstracts: S01: New insights into fetal masculinisation disorders and the implications for endocrine disruptor effects

Becoming a male is ultimately determined by androgen-induced masculinisation. Disorders of this, resulting in hypospadias (abnormal penis development) or cryptorchidism (undescended testes), are common disorders in humans. Together with adult onset disorders (low sperm counts, testis germ cell cancer) hypospadias and cryptorchidism may constitute a testicular dysgenesis syndrome (TDS) in humans, with a proposed common fetal origin that may involve deficiencies in androgen production or action. As animal evidence has shown that anti-androgenic endocrine disruptors (EDs) can induce TDS-like effects, concern has grown regarding their potential involvement in human TDS disorders. We have developed a rat model of TDS, in which we have begun to dissect the fetal mechanisms that underlie TDS disorders. Using another rat model in which androgen action is blocked by the anti-androgen flutamide, we have recently identified a ‘male programming window’, which available evidence suggests occurs also in the human fetus (between ∼8 and 12 week's gestation). Androgens must act within this programming window to set up later correct development of the reproductive tract and genitalia. Surprisingly, androgen-driven (abnormal) masculinisation of females is confined to the same programming window as for normal males. Blocking androgen action only within the programming window induces hypospadias and cryptorchidism and alters penile length in males; these all correlate with reduction in anogenital distance (AGD), which provides a non-invasive, lifelong read-out of androgen exposure in the programming window (but not later in gestation). As AGD is measurable in human newborns/neonates, it may predict adult onset TDS disorders and provide clinically important insights into reproductive tract masculinisation and its disorders. With this new understanding we have also begun to explore the role and timescale of effects of androgens in regulation of penile development and size. We show in the rat, that AGD predicts fetal and adult testis size (and thus sperm production), but the mechanism underlying this is unknown as it is not explained by effects on Sertoli cell number, as androgen regulation of Sertoli cell proliferation extends beyond the ‘male programming window’ (when AGD is determined); this relationship is of potential importance in the context of fetal origins of low sperm counts in humans. These findings strongly support the view that deficient fetal androgen action is a key feature of TDS. We postulate that delayed onset of fetal testosterone production may also be important in TDS. These new findings have considerable implications with regard to the time-window of susceptibility of the fetal male to disruption by EDs.

Maternal adrenal hormone secretion mediates behavioural alterations induced by prenatal stress in male and female rats

Prenatal stress in rats has been shown to impair the regulation of the hypothalamic pituitary adrenal (HPA) axis and predispose to anxiogenic and depressive-like behaviour. In a previous study, abolition of excess corticosterone (COR) release during stress by maternal adrenalectomy prevented the dysregulation of the HPA axis. In the present study, we determined whether excess maternal COR is also responsible for the alterations in offspring behaviour. Pregnant Wistar rats were adrenalectomized or sham-operated on day 11 of gestation and subjected once daily to mild restraint for 30 min on days 14–21 of gestation. An undisturbed group of pregnant females served as controls. All experiments were performed in male and female offspring. Pup weight and anogenital distance of males were measured after birth; anxiogenic behaviour was assessed in the elevated plus maze (EPM) at the age of 5 weeks and spatial memory in the Morris water maze in littermates at 3–4 months. Prenatally stressed (PS) males did not show a reduction in anogenital distance, and their increase in anxiogenic behaviour in the EPM was less than that in PS females. On the other hand, impairment of spatial learning was only seen in PS males. Both the anxiogenic behaviour of PS males and females and the learning deficit in males were completely abolished by adrenalectomy. These data show that excess stress-induced COR can alter the programming of the foetal brain and predispose it to alterations in behaviour that are gender specific.

Could neonatal testosterone replacement prevent alterations induced by prenatal stress in male rats?

The present study was designed to examine whether testosterone replacement is able to prevent some effects of maternal restraint stress — during the period of brain sexual differentiation — on endocrine system and sexual behavior in male rat descendants. Pregnant rats were exposed to restraint stress for 1 h/day from gestational days 18 to 22. At birth, some male pups from these stressed rats received testosterone propionate. The neonatal testosterone replacement was able to prevent the reduction in anogenital distance at 22 days of age observed in pups from stressed pregnant rats as well as prevents the decrease in testosterone levels during the adulthood of these animals. Testosterone replacement in these males also presented an improvement in sexual performance. In this way, testosterone replacement probably through increasing neonatal level of this hormone was able to prevent the later alterations caused by the prenatal stress during the period of brain sexual differentiation.

Guest Editorial: Phthalate Exposure during Pregnancy and Lower Anogenital Index in Boys: Wider Implications for the General Population?

Guest Editorial: Phthalate Exposure during Pregnancy and Lower Anogenital Index in Boys: Wider Implications for the General Population?

Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat.

Fenitrothion [0,0-dimethyl-O-(4-nitro-m-tolyl) phosphorothioate] is an organophosphate insecticide that has been shown to have antiandrogenic activity using in vitro and in vivo screening assays. Studies were performed to evaluate the ability of fenitrothion to disrupt androgen-dependent sexual differentiation in the male rat. Pregnant Crl:CD(SD)BR rats were administered fenitrothion by gavage at 0, 5, 10, 15, 20, or 25 mg/kg/day ( n = 6-11/group) from gestation day (GD) 12 to 21. Maternal toxicity was observed in the dams treated with 20 and 25 mg fenitrothion/kg/day based on muscle tremors and decreases in body weight gain from GD 12 to 21. Fetal death was increased in the 20 and 25 mg/kg/day exposure groups, as evidenced by a decrease in the proportion of pups born alive. Androgen-mediated development of the reproductive tract was altered in male offspring exposed in utero to maternally toxic levels of fenitrothion (25 mg/kg/day), as evidenced by reduction in anogenital distance on postnatal day (PND) 1 and retention of areolae on PND 13. However, these effects were only transient, and there were no indications of abnormal phenotypes or development of androgen-dependent tissues on PND 100. At the dose levels evaluated in this study, fenitrothion was only weakly antiandrogenic in vivo compared with other androgen receptor antagonists such as flutamide, linuron, and vinclozolin. Based on observed fetotoxicity at 20 mg/kg/day, the lowest observed adverse effect level (LOAEL) for developmental effects can be lowered from 25 to 20 mg/kg/day.