Male Anogenital Distance Research Articles

7849 The EDC Mixture “NeuroMix” Has Developmental, Anxiolytic, And Obesogenic Effects In Rats

Abstract Disclosure: E. Morales-Grahl: None. L. M. Thompson: None. E.N. Hilz: None. A.C. Gore: None. Humans are exposed to a variety of endocrine disrupting chemicals (EDCs) on a daily basis through drinking water, food, and contact with everyday objects. Safety guidelines aim to keep exposure to individual EDCs below certain levels but don’t take into account the potentially unpredictable effects of simultaneous exposure to multiple EDCs. Furthermore, EDCs may exhibit non-monotonic dose responses. The present study assessed the effect of an EDC mixture at three different doses on two sets of behaviors implicated in EDC research: anxiety, and high fat and sugar consumption. Beginning in early pregnancy and until pups were weaned at day 21, Sprague Dawley rats were fed daily with NeuroMix, our lab’s EDC mixture containing 9 common industrial chemicals, at three doses (0.1x, 1x, 10x). The base dose (1x) contained EDCs below the no observed adverse effect level in humans (NOAEL). In adulthood, the female and male offspring were then subjected to an open field and light/dark test, followed by a high fat and high sucrose preference test. 0.1x NeuroMix exposure advanced the timing of vaginal opening in female offspring, while all doses increased anogenital distance in males, marking differences in sexual development. 1x NeuroMix exposure in male rats increased their time in the open field corners and their time immobile in the corners, indicating higher anxiety. No difference was seen in sucrose preference. Female rats exposed to 1x NeuroMix showed a higher preference for a high-fat diet, and gained more weight compared to the controls while on this diet. These findings demonstrate that a mixture of human-relevant chemicals, when administered perinatally to rats, have developmental, anxiogenic and obesogenic effects in a sexually dimorphic manner. We also show a non-monotonic response to our chemical mixture, with the 1x dose showing greater effects than the 10x dose. Overall, we provide evidence that even at levels considered safe for individual EDCs, there are adverse developmental and behavioral effects of EDCs when combined. Grant support: R35 ES035024, F32 ES034257. Presentation: 6/1/2024

Cypermethrin Toxicity-induced Infertility: An Assessment of Longitudinal Transfer of Protective Potentials of Date Fruit Extracts in Wister Rats.

There is a potential for longitudinal and horizontal transfer of the cytotoxic effects of cypermethrin, and evidence suggests that date fruit extracts can ameliorate these cytotoxic effects. Thus, the current study evaluated female Wister rats' fertility and pup parameters after mating males treated with cypermethrin and date fruit (Phoenix dactylifera) extract. Adult Wistar rats (n = 74; 140 - 207 g) were used to conduct this experiment in two phases: The first phase was a single dose toxicity study (n = 18), while the second phase (n = 56) evaluated female Wister rats' reproductive and pup development parameters after mating with male Wistar rats exposed to date fruit extract and cypermethrin. Male rats were distributed randomly into four groups (n = 7 per group) and administered distilled water (Group I); 60 mg/kg cypermethrin (Group II) to simulate infertility; 250 mg/kg date fruit extract alone (Group III); and date fruit extracts plus 60 mg/kg cypermethrin (Group IV). Treated males were then mated with healthy female rats at a ratio of 1:1. Successfully mated females were appraised for conception and pregnancy rates, gestation length, litter size, and weights at birth, and on days 5, 10, 15, and 21 post-partum, an anogenital distance at day-20, sex ratio, and age of pup when hair growth was evident. Significant differences (p < 0.05) were observed in litter size and male anogenital distance but not in reproductive performance. Also, the study demonstrated that date fruit extract skewed the sex ratio of offspring to more female than male pups when female Wister rats were mated with males that received date fruit extracts alone or cypermethrin and date fruit extracts. The body weight gain did not differ significantly between the control and cypermethrin-treated groups. This evidence suggested a longitudinal transfer of the protective effects of date fruit extracts following a weekly exposure of male rats to cypermethrin at 60 mg/kg.

PMON251 Loss of Constitutive Androstane Receptor, CAR reduces the serum androgens levels and alters reproductive function

Abstract The nuclear receptor, Constitutive Androstane Receptor (CAR/NR1i3) is extensively known for its detoxification function in the liver. We have identified the role of CAR in regulating androgen levels and regulating testis function. Although the inactivation of CAR by the sex hormone axis via androstanol has been known for decades, its role in regulating reproduction has not been addressed. On examining the WT mice and the global CARKO mice, we found that CARKO mice have reduced serum testosterone and androstanol levels. Further, the levels of the steroidogenic gene, Srd5a1 was significantly reduced. This result correlated well with reduced anogenital distance in males, increased anxiety and subfertility. However, we did not find differences in the expression of detoxification genes in the CARKO testis. Histological analysis of the testis of the CARKO mice revealed abnormal Leydig cells and Oil Red O staining showed lipid accumulation in the Leydig cell island. We are currently examining the effect of the reduced androgens in the hypothalamic-pituitary-gonadal axis in the CARKO mice model. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The anti-androgenic fungicide triticonazole induces region-specific transcriptional changes in the developing rat perineum and phallus

Intrauterine exposure to endocrine disrupting chemicals can interfere with male reproductive development. This can lead to male reproductive disorders such as hypospadias, cryptorchidism and reduced fertility, as well as shorter anogenital distance (AGD) – a biomarker for incomplete androgen-dependent fetal masculinization. However, it remains challenging to predict adverse in vivo outcomes based on in vitro effect patterns for many chemicals. This is a challenge for modern toxicology, which aims to reduce animal testing for chemical safety assessments. To enable the transition towards higher reliance on alternative test methods, we need to better map underlying mechanisms leading to adverse effects. Herein, we have analyzed the transcriptome of the perineum and phallus of male fetal rats and defined the impacts of exposure to an anti-androgenic fungicide, triticonazole. Previously we have shown that developmental exposure to triticonazole can induce short male AGD, but without a marked effect on the transcriptome of the fetal testes. In contrast, we report here significant changes to the transcriptional landscape of the perineum and phallus, including regional differences between these adjacent tissues. This highlights the importance of analyzing the correct tissue when characterizing mechanisms of complex in vivo effect outcomes. Our results provide a rich resource for the spatiotemporal gene networks that are involved in the development of male external genitalia, and that can be disrupted upon exposure to chemicals that prevent normal masculinization of the perineum and phallus. Such data will be critical in the development of novel alternative test methods to determine the endocrine disrupting potential of existing and emerging chemicals.

N/C Interactions Are Dispensable for Normal In Vivo Functioning of the Androgen Receptor in Male Mice.

The androgen receptor (AR) plays a central role in the development and maintenance of the male phenotype. The binding of androgens to the receptor induces interactions between the carboxyterminal ligand-binding domain and the highly conserved 23FQNLF27 motif in the aminoterminal domain. The role of these so-called N/C interactions in AR functioning is debated. In vitro assays show that mutating the AR in the 23FQNLF27 motif (called ARNoC) attenuates the AR transactivation of reporter genes, has no effect on ligand binding, but does affect protein-protein interactions with several AR coregulators. To test the in vivo relevance of the N/C interaction, we analyzed the consequences of the genomic introduction of the ARNoC mutation in mice. Surprisingly, the ARNoC/Y mice show a normal male development, with unaffected male anogenital distance and normal accessory sex glands, male circulating androgen levels, body composition, and fertility. The responsiveness of androgen target genes in kidney, prostate, and testes was also unaffected. We thus conclude that the N/C interactions in the AR are not essential for the development of a male phenotype under normal physiological conditions.

In Utero Exposure to a Mixture of the Perfluoroalkyl-Isopropyl Pesticide Pyrifluquinazon With Dibutyl Phthalate Cumulatively Disrupts Male Rat Reproductive Development via Different Mechanisms of Action.

Administration of individual chemicals and mixtures during sexual differentiation that disrupt the androgen signaling pathway can induce reproductive abnormalities in male rats. In this study, we coadministered the heptafluoroisopropyl pesticide pyrifluquinazon (PFQ), and dibutyl phthalate (DBP) to pregnant rats during sexual differentiation of the reproductive tract. Both chemicals have been shown to disrupt reproductive tract differentiation in a dose-related manner reducing male anogenital distance, permanently reducing androgen-dependent tissue weights and sperm counts, and inducing reproductive malformations in male offspring, albeit by different mechanisms of action that converge downstream in the androgen signaling pathway on a common key event. Rats were orally dosed from gestation days 14-18 with dilutions of PFQ and DBP at 0%, 12.5%, 25%, 50%, 75%, and 100% of the top dose (100 mg/kg PFQ and 750 mg/kg DBP). The mixture ratio was selected such that each chemical would contribute equally to multiple effects on the male offspring reproductive tract and the dose range was designed to determine if the mixture produced additive effects predicted by dose addition (DA) or response addition (RA) models, or whether significant interactions occurred. Observed data were compared with DA and RA model predictions. As hypothesized, the mixture reduced F1 male anogenital distance, reproductive organ weights and sperm counts and induced hypospadias with DA consistently providing a better prediction of the observed effects than RA. These results support our hypothesis that chemicals that disrupt the androgen signaling pathway induce dose-additive male reproductive abnormalities regardless of the specific mechanism of action.

On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies.

Areola/nipple retention (NR) is an established biomarker for an anti-androgenic mode of action in rat toxicity studies. It is a mandatory measurement under several OECD test guidelines and is typically assessed in combination with anogenital distance (AGD). Both NR and AGD are considered retrospective biomarkers of insufficient androgen signaling during the masculinization programming window in male fetuses. However, there are still aspects concerning NR as a biomarker for endocrine disruption that remains to be clarified. For instance, can NR be regarded a permanent adverse effect? Is it a redundant measurement if AGD is assessed in the same study? Is NR equally sensitive and specific to anti-androgenic chemical substances as a shortening of male AGD? In this review we discuss these and other aspects concerning the use of NR as a biomarker in toxicity studies. We have collected available literature from rat toxicity studies that have reported on NR and synthesized the data in order to draw a clearer picture about the sensitivity and specificity of NR as an effect biomarker for an anti-androgenic mode of action, including comparisons to AGD measurements. We carefully conclude that NR and AGD in rats for the most part display similar sensitivity and specificity, but that there are clear exceptions which support the continued assessment of both endpoints in relevant reproductive toxicity studies. Available literature also support the view that NR in infant male rats signifies a high risk for permanent nipples in adulthood. Finally, the literature suggests that the mechanisms of action leading from a chemical stressor event to either NR or short AGD in male offspring are overlapping with respect to canonical androgen signaling, yet differ with respect to other mechanisms of action.

Maternal exposure to perfluoroalkyl chemicals and anogenital distance in the offspring: A Faroese cohort study

Exposure to perfluoroalkyl substances (PFASs) has in some studies been associated with reduced anogenital distance (AGD) in newborns as a sensitive indicator of prenatal anti-androgenic exposure. The aim of this study was to investigate the association between maternal PFAS exposure and offspring AGD in a population with wide ranges of PFAS exposures.Participants were recruited in the Faroe Islands in 2007–2009, and information on AGD and PFAS exposure was obtained from 463 mother-infant pairs. Perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) were measured in maternal serum. Data were analyzed using multiple linear regression analysis adjusted for birth weight, child age at examination, parity, and maternal education level.Among boys, higher maternal serum concentrations of PFOA, PFOS, PFNA and PFDA were significantly associated with a longer AGD, both with the exposure entered as a continuous variable and as quartiles. Boys in the highest quartile of PFOA, PFOS, PFNA and PFDA exposure had an increase in AGD of 1.2 mm (95 % CI 0.1;2.2), 1.3 mm (95 % CI 0.3;2.3), 1.0 mm (95 % CI 0.0:2.0) and 1.3 mm (95 % CI 0.3;2.4), respectively, when compared to boys in the lowest quartile of exposure (p < 0.05). No significant association was found between male AGD and PFHxS. No association was found for girls.In conclusion, elevated maternal exposure to major PFASs was significantly associated with a longer AGD in boys. No significant associations were found among girls, thus suggesting a sex-dimorphic effect of PFAS exposure.

Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats

Clotrimazole is a non-prescription and broad-spectrum antifungal drug sold under brand names such as Canesten® and Lotrimin®. It is used to treat different types of fungal infections, from oral thrush to athlete's foot and vaginal mycosis. The level of exposure to clotrimazole is uncertain, as the exact usage amongst self-medicating patients is unclear. Recent studies have raised potential concern about the unsupervised use of clotrimazole during pregnancy, especially since it is a potent inhibitor of CYP enzymes of the steroidogenesis pathway. To address some of these concerns, we have assessed the effects of intrauterine exposure to clotrimazole on developing rat fetuses. By exposing pregnant rats to clotrimazole 25 or 75 mg/kg bw/day during gestation days 7–21, we obtained internal fetal concentrations close to those observed in humans. These in vivo data are in strong agreement with our physiologically-based pharmacokinetic (PBK)-modelled levels. At these doses, we observed no obvious morphological changes to the reproductive system, nor shorter male anogenital distance; a well-established morphometric marker for anti-androgenic effects in male offspring. However, steroid hormone profiles were significantly affected in both maternal and fetal plasma, in particular pronounced suppression of estrogens was seen. In fetal testes, marked up-concentration of hydroxyprogesterone was observed, which indicates a specific action on steroidogenesis. Since systemic clotrimazole is rapidly metabolized in humans, relevant exposure levels may not in itself cause adverse changes to the reproductive systems. Its capacity to significantly alter steroid hormone concentrations, however, suggests that clotrimazole should be used with caution during pregnancy.

Prenatal exposure to bisphenols affects pregnancy outcomes and offspring development in rats

The objective of this study was to evaluate the effects of gestational exposure to low doses of bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF) on pregnancy outcomes and offspring development. Pregnant Sprague-Dawley rats were orally dosed with vehicle, 5 μg/kg body weight (BW)/day of BPA, BPS and BPF, or 1 μg/kg BW/day of BPF on gestational days 6–21. Pregnancy and gestational outcomes, including number of abortions and stillbirths, were monitored. Male and female offspring were subjected to morphometry at birth, followed by pre- and post-weaning body weights, post-weaning food and water intakes, and adult organ weights. Ovarian follicular counts were also obtained from adult female offspring. We observed spontaneous abortions in over 80% of dams exposed to 5 μg/kg of BPF. BPA exposure increased Graafian follicles in female offspring, while BPS and BPF exposure decreased the number of corpora lutea, suggesting reduced ovulation rates. Moreover, BPA exposure increased male kidney and prostate gland weights, BPF decreased epididymal adipose tissue weights, and BPS had modest effects on male abdominal adipose tissue weights. Prenatal BPS exposure reduced anogenital distance (AGD) in male offspring, suggesting possible feminization, whereas both BPS and BPA induced oxidative stress in the testes. These results indicate that prenatal exposure to BPF affects pregnancy outcomes, BPS alters male AGD, and all three bisphenols alter certain organ weights in male offspring and ovarian function in female offspring. Altogether, it appears that prenatal exposure to BPA or its analogues can induce reproductive toxicity even at low doses.

Prenatal paraben exposure and anogenital distance and reproductive hormones during mini-puberty: A study from the Odense Child Cohort

BackgroundParabens are added to personal care products as antimicrobial preservatives. They have been suggested to have endocrine disrupting abilities. Prenatal exposure to parabens has been associated with reproductive endpoints including reduced male anogenital distance (AGD, distance from anus to genitals), which is sensitive to prenatal anti-androgenic exposure. ObjectivesTo study the associations between maternal paraben concentrations in second trimester urine and AGD and reproductive hormone concentrations at 3 months of age in offspring. MethodsPregnant women from Odense, Denmark were included in early pregnancy from 2010 to 12, and their children are being followed up. Fasting spot urine samples from 536 pregnant women were analyzed for methylparaben (MeP), ethyl-paraben (EtP), iso-propylparaben (i-PrP), n-propylparaben (n-PrP), n-butylparaben (n-BuP) and benzylparaben (BzP) by liquid chromatography tandem mass spectrometry and thereafter osmolarity adjusted. Three months after expected date of birth, AGD was measured in 452 children, and serum concentrations of follicle stimulating hormone (FSH), luteinizing (LH), testosterone, dehydroepiandrosterone-sulphate (DHEAS), androstenedione and 17-hydroxyprogesterone (17-OHP) were measured in 198 children. Maternal paraben exposure was categorized into tertiles or below and above level of detection, and sex-stratified multiple linear regression analyses were performed with AGD or reproductive hormones as outcomes. ResultsMost pregnant women had low concentrations of parabens in urine, but 10% exceeded the threshold for adverse estrogenic effects. Higher maternal paraben exposure was associated with shorter AGD in male offspring and longer AGD in girls, although only significant for MeP in boys. In addition, FSH, LH, DHEAS, 17-OHP concentrations were lower in girls with high prenatal paraben exposure, whereas no consistent pattern was found in boys. DiscussionThe endocrine disrupting abilities of parabens may affect humans at vulnerable time periods during development, which may have long term impact on reproductive function. This is the first study to find these associations in girls and our findings need confirmation.

Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3′Digital Gene Expression (3′DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

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.

Androgens and the masculinization programming window: human-rodent differences.

Human male reproductive disorders are common and may have a fetal origin — the testicular dysgenesis syndrome (TDS) hypothesis. In rats, experimentally induced TDS disorders result from disruption of fetal androgen production/action specifically in the masculinization programming window (MPW). MPW androgen action also programs longer anogenital distance (AGD) in male versus female rats; shorter male AGD is correlated with risk and severity of induced TDS disorders. AGD thus provides a lifelong, calibrated readout of MPW androgen exposure and predicts likelihood of reproductive dysfunction. Pregnant rat exposure to environmental chemicals, notably certain phthalates (e.g. diethyl hexl phthalate, DEHP; dibutyl phthalate, DBP), pesticides or paracetamol, can reduce fetal testis testosterone and AGD and induce TDS disorders, provided exposure includes the MPW. In humans, AGD is longer in males than females and the presumptive MPW is 8–14 weeks’ gestation. Some, but not all, epidemiological studies of maternal DEHP (or pesticides) exposure reported shorter AGD in sons, but this occurred at DEHP exposure levels several thousand-fold lower than are effective in rats. In fetal human testis culture/xenografts, DEHP/DBP do not reduce testosterone production, whereas therapeutic paracetamol exposure does. In humans, androgen production in the MPW is controlled differently (human chorionic gonadotrophin-driven) than in rats (paracrine controlled), and other organs (placenta, liver, adrenals) contribute to MPW androgens, essential for normal masculinization, via the ‘backdoor pathway’. Consequently, early placental dysfunction, which is affected by maternal lifestyle and diet, and maternal painkiller use, may be more important than environmental chemical exposures in the origin of TDS in humans.

Prenatal perfluoroalkyl substances and newborn anogenital distance in a Canadian cohort

Exposure to the man-made chemicals perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexanesulfonate (PFHxS) is widespread. These perfluoroalkyl substances (PFASs) have been associated with androgenic endocrine-disrupting properties; however, the evidence is equivocal and few human studies have examined the association between prenatal exposure to PFASs and markers of androgenic endocrine disruption such as changes in anogenital distance (AGD).In the MIREC cohort, PFOA, PFOS and PFHxS were analyzed in first trimester maternal plasma. AGD was measured in 205 male and 196 female newborns. The change in estimate procedure was used to identify confounders by sex and AGD in multiple linear regression models.Geometric mean plasma concentrations (95% CI) for PFOA, PFOS and PFHxS were 1.71 (1.61, 1.81), 4.40 (4.18, 4.64) and 1.15 (1.06, 1.25) μg/L, respectively. A one-unit increase in natural log transformed PFOA was associated with a 1.36 mm (95% CI 0.30, 2.41) increase in anoscrotal distance, adjusting for household income, active smoking status during pregnancy and gestational age. However, when examined by quartiles, a non-monotonic pattern was observed with wide confidence intervals. No consistent patterns were observed between maternal PFAS concentrations and female AGDs.This study found no clear evidence that maternal plasma concentrations of PFOS, PFOA or PFHxS were associated with shorter infant anogenital distance in males or any change in AGD in females. Whether the positive association observed between longer anoscrotal distance and PFOA is real or would have any long-lasting effect on the reproductive health of males is unknown and needs to be investigated further.

Anogenital distance as a measure of male competitive ability in Rwenzori Angolan colobus.

Anogenital distance (AGD) is positively correlated to fetal androgen exposure and developmental masculinization in mammals. Independent of overall body size, AGD shows a strong positive correlation with male fertility and in rodents, AGD is a good indicator of male competitive ability and is associated with female choice. We hypothesized that AGD will also predict male competitive ability in non-human primates. To test this, we measured AGD noninvasively with a parallel laser in a wild population of Angolan colobus monkeys (Colobus angolensis ruwenzorii) in Uganda and correlated to it to their social structure. C. angolensis ruwenzorii form a multilevel society with both one-male/multifemale units (OMUs) and multimale/multifemale units (MMUs). We compared AGD in males from five OMUs and six MMUs and related it to their fecal androgen metabolite concentrations, dominance rank and body size, and to the number of females in their unit. Males in OMUs had greater access to females, so were predicted to have longer AGDs, but this was not found. AGD also did not correlate overall with mean fecal androgen metabolites in MMUs. However, AGD was correlated with dominance rank in MMUs, demonstrating that higher-ranking males in these multimale units had longer AGDs. Body size did not show the same relationship with dominance rank, suggesting that male rank was not just a reflection of absolute male size. Our findings indicate that AGD predicts male competitive ability in this species and that it may be a useful correlate throughout the non-human primates. These results also support the idea that prenatal androgen exposure increases the likelihood of the expression of behaviors that maintain high dominance rank.

In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole

Azoles are effective antifungal agents used in both medicine and agriculture. They typically work by inhibiting cytochrome P450 enzymes, primarily CYP51 of the ergosterol biosynthesis pathway, thus damaging the fungal cell membrane. However, apart from their desired antifungal properties, several azoles also exhibit endocrine disrupting properties in mammals, both in vitro and in vivo. Here, we have tested two currently used agricultural azole fungicides, triticonazole and flusilazole, for their in vitro anti-androgenic activity and potential effects on reproductive parameters. Both fungicides showed strong androgen receptor (AR) antagonism and disruption of steroid biosynthesis in vitro. Following gestational exposure to flusilazole (15 or 45 mg/kg bw/day) or triticonazole (150 or 450 mg/kg bw/day) in time-mated Sprague Dawley rats, triticonazole induced shorter male anogenital distance (AGD). Flusilazole exposure did not affect the AGD, but altered fetal male blood hormone profile, with increased androstenedione and decreased estrone levels. Flusilazole and triticonazole have dissimilar effects on reproductive parameters in vivo, but both show endocrine disrupting activities.

Do stressful life events during pregnancy modify associations between phthalates and anogenital distance in newborns?

Anogenital distance (AGD) has been used as a marker of fetal androgen action to identify endocrine disrupting chemicals. A US study (TIDES) has reported that the association between some phthalates and reduced AGD in males was only apparent in sons of mothers reporting no stressful life events (SLEs) during pregnancy. The objective of the current study was to examine the potential modifying effect of SLEs and their subjective impact on associations between prenatal phthalates and AGD. First trimester urines from the MIREC Study were analysed for phthalate metabolites and AGD was measured in neonates. Post-delivery, the women answered questions on SLEs during the pregnancy. Women reporting 1 or more SLEs during pregnancy were considered a “higher stressor” group, whereas women reporting no SLEs or who reported a SLE that was perceived as not at all stressful were considered a “lower stressor” group. Multivariable linear regression models were fit stratified by stressor group.Maternal stressor, AGD and phthalates results were available for 153 females and 147 males. A summary measure of androgen-disrupting phthalates (Σ AD) was associated with significantly longer AGDs in females from the higher stressor group. These effect sizes were increased when the perceived impact was restricted to moderately or very much stressful. In males, all phthalates were associated with longer anopenile distance (APD), regardless of stressor group; however, higher Σ AD was associated with significantly longer APD in the lower stressor group. In contrast to the TIDES study, we did not observe shorter AGDs in male infants prenatally exposed to di-(2-ethylhexyl) phthalates, regardless of maternal stressor level.In conclusion, we were unable to replicate the findings of the TIDES study, but did find some evidence that prenatal SLEs may modify associations between phthalates and female AGD. Further research with other populations and measures of prenatal stress may shed more light on whether prenatal stress is an important effect modifier of associations between phthalates (or other chemicals) and anogenital distance.

Fetal anogenital distance using ultrasound.

ObjectiveThis study measured anogenital distance (AGD) during late second/early third trimester of pregnancy to confirm previous findings that AGD can be measured noninvasively in the fetus using ultrasound and further showed differences in reference ranges between populations.MethodTwo hundred ten singleton pregnancies were recruited at the Rosie Hospital, Cambridge, UK. A 2D ultrasound was performed between 26 and 30 weeks of pregnancy. AGD was measured from the centre of the anus to the base of the scrotum in males and to the posterior convergence of the fourchette in females.ResultsA significant difference in AGD between males and females (P < .0001) was found, replicating previous results with a significant correlation between estimated fetal weight (EFW) and AGD in males only (P = .006). A comparison of AGD using reference data from an Israeli sample (n = 118) and our UK sample (n = 208) showed a significant difference (P < .0001) in both males and females, after controlling for gestational age (GA).ConclusionOur results confirm that AGD measurement in utero using ultrasound is feasible. In addition, there are strong sex differences, consistent with previous suggestions that AGD is influenced by prenatal androgen exposure. AGD lengths differ between the UK and Israel; therefore, population‐specific normative values may be required for accurate clinical assessments.

The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on development and endocrine system

BackgroundGlyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential effects on the endocrine system The present pilot study examine whether exposure to GBHs at the dose of glyphosate considered to be “safe” (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero life, affect the development and endocrine system across different life stages in Sprague Dawley (SD) rats.MethodsGlyphosate alone and Roundup Bioflow, a commercial brand of GBHs, were administered in drinking water at 1.75 mg/kg bw/day to F0 dams starting from the gestational day (GD) 6 (in utero) up to postnatal day (PND) 120. After weaning, offspring were randomly distributed in two cohorts: 8 M + 8F/group animals belonging to the 6-week cohort were sacrificed after puberty at PND 73 ± 2; 10 M + 10F/group animals belonging to the 13-week cohort were sacrificed at adulthood at PND 125 ± 2. Effects of glyphosate or Roundup exposure were assessed on developmental landmarks and sexual characteristics of pups.ResultsIn pups, anogenital distance (AGD) at PND 4 was statistically significantly increased both in Roundup-treated males and females and in glyphosate-treated males. Age at first estrous (FE) was significantly delayed in the Roundup-exposed group and serum testosterone concentration significantly increased in Roundup-treated female offspring from the 13-week cohort compared to control animals. A statistically significant increase in plasma TSH concentration was observed in glyphosate-treated males compared with control animals as well as a statistically significant decrease in DHT and increase in BDNF in Roundup-treated males. Hormonal status imbalances were more pronounced in Roundup-treated rats after prolonged exposure.ConclusionsThe present pilot study demonstrate that GBHs exposure, from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. In particular, it was associated with androgen-like effects, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in female.