Decrease In Anogenital Distance Research Articles

Phenotypic and genetic relationships among anogenital distance, anti-Müllerian hormone, and in vitro embryo production in Gyr dairy cattle

Anti-Müllerian hormone (AMH) concentration and number of recovered oocytes (ROOC) are phenotypic parameters associated with in vitro embryo production (IVEP). More recently, anogenital distance (AGD) has been proposed as a proxy for fertility in dairy cattle that is easy to collect at a low cost. The aim of this study was to characterize the AGD and its phenotypic and genetic associations with AMH and IVEP in Bos indicus Gyr dairy cattle. The hypothesis was that the number of ROOC, in vitro-produced embryos, and AMH concentration would increase as the AGD decreases. From July to December 2021, a single morphometrical measurement of AGD was collected in 552 donors from 6 herds in Brazil. A subset of donors had AMH assayed on the same day. Only ovum pick-up events that occurred up to 12 mo preceding and 7 mo succeeding the AGD measurement were used to assess the association between AGD, AMH, and IVEP. Thus, 472 donors (1,551 ovum pick-up events and 140 donors with AMH) were considered in the analysis. A raw average was calculated for each individual donor's ROOC, viable oocytes, total produced embryos, viability rate, and embryo rate (defined as total produced embryos/viable oocytes). Comparisons were conducted within the age categories of 3 to <6 yr or 6 to <10 yr. Phenotypic associations were performed in SAS software (SAS Institute Inc., Cary, NC). Genetic correlations were estimated using the BLUPF90 family of programs. The AGD (128.7 mm ± 14; mean ± standard deviation) had a normal distribution and was highly variable (83 to 172 mm) among the Gyr population. Our experimental hypothesis was partially supported by a phenotypic association of a greater number of total produced embryos (R2 = 0.023) as AGD decreased. Our results failed to support an increase in AMH concentration along with a decrease in AGD. In addition, positive and low genetic correlations were observed between AGD and viable oocytes (r = 0.08), and embryo rate (r = 0.20). A greater number of viable oocytes and embryos were observed in donors in the high compared with intermediate and low ROOC categories within both age categories. The age interval of 3 to <6 yr showed a greater number of recovered and viable oocytes for the high AMH compared with the low category, but no differences were observed among the AGD categories. In summary, for the Gyr breed, AGD was phenotypically inversely associated with a quantity-related parameter, such as the total number of produced embryos. In contrast, AGD showed a low genetic correlation with qualitative-related outcomes such as viable oocytes and embryo rate. Further studies should be performed to validate these retrospective analyses and to better understand the association between AGD and IVEP.

Paternal low-dose benzo(a)pyrene exposure in rats impairs sexual development and fertility of the paternal lineage in F2 generation: A transgenerational study

The field of Paternal Origins of Health and Disease (POHaD) is highly relevant but remains under-explored. The F2 generation from males indirectly exposed (F1 - via germ cells) to benzo(a)pyrene (BaP), named PF2, was investigated in this study under parameters of sexual development and reproductive performance of male and female rats. Male Wistar rats (F0) were exposed to BaP (0.1 µg/kg/day) for 31 consecutive days (gavage) during prepuberty. The F0 rats were mated with untreated females to produce male offspring (F1), which were exposed to BaP via germ cells. The F1 males were later mated with untreated females to obtain the PF2 generation, which was the focus of our investigation. Our findings showed that PF2 males exhibited a decrease in anogenital distance, fertility potential, testosterone levels, and type A sperm. Meanwhile, PF2 females had an earlier vaginal opening, lower lordosis scores, and decreased fertility. Furthermore, changes in the histomorphology of the testis/epididymis and ovary/uterus were observed. The repercussions of the PF2 generation indicate that these animals showed losses in both sexual development and fertility potential, and we can conclude that this damage remained due to paternal transgenerational inheritance caused by a low dose of BaP.

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.

Oral exposure of rats to dienestrol during gestation and lactation: Effects on the reproductive system of male offspring

This study was aimed at determining whether dienestrol (DIES) affects reproduction in male offspring of rats following oral maternal exposure during gestation and lactation. Pregnant rats were treated from GD 6 to PND 21. Animals received 0 (control-vehicle), 0.75, 1.5, 3.12, 6.25, 12.5, 50, 75 μg/kg bw/d of DIES. A control group -without vehicle-was also included. High DIES concentrations caused abortions at 75 and 50 μg/kg bw/d, while at 12.5 μg/kg bw/d had still miscarriages. Ten male rats per group were kept alive until PND 90 to ensure sexual maturity. Body and organ weights, anogenital distance (AGD) at PNDs 21 and 90, biochemical and sperm parameters like motility, viability, morphology, spermatozoa and resistant spermatid counts, and histopathology for sexual organs and liver were determined. An increase in organ weight (liver and sexual organs) and a decrease in AGD due to vehicle were found. A reduction of sperm motility and viability, and an increase of abnormal sperm morphology were caused by DIES, which provoked a dose-dependent prostatitis. Maternal exposure to DIES induced toxicity on the reproductive system of the male offspring, which could affect the capacity of fertilization.

Perfluorohexane Sulfonate (PFHxS) and a Mixture of Endocrine Disrupters Reduce Thyroxine Levels and Cause Antiandrogenic Effects in Rats.

The developmental toxicity of perfluorohexane sulfonate (PFHxS) is largely unknown despite widespread environmental contamination and presence in human serum, tissues and milk. To thoroughly investigate PFHxS toxicity in developing rats and to mimic a realistic human exposure situation, we examined a low dose close to human relevant PFHxS exposure, and combined the dose-response studies of PFHxS with a fixed dose of 12 environmentally relevant endocrine disrupting chemicals (EDmix). Two reproductive toxicity studies in time-mated Wistar rats exposed throughout gestation and lactation were performed. Study 1 included control, two doses of PFHxS, and two doses of PFHxS + EDmix (n = 5-7). Study 2 included control, 0.05, 5, or 25 mg/kg body weight/day PFHxS, EDmix-only, 0.05, 5, or 25 mg PFHxS/kg plus EDmix (n = 13-20). PFHxS caused no overt toxicity in dams and offspring but decreased male pup birth weight and slightly increased liver weights at high doses and in combination with the EDmix. A marked effect on T4 levels was seen in both dams and offspring, with significant reductions from 5 mg/kg/day. The EDmix caused antiandrogenic effects in male offspring, manifested as slight decreases in anogenital distance, increased nipple retention and reductions of the weight of epididymides, ventral prostrate, and vesicular seminalis. PFHxS can induce developmental toxicity and in addition results of the co-exposure studies indicated that PFHxS and the EDmix potentiate the effect of each other on various endpoints, despite their different modes of action. Hence, risk assessment may underestimate toxicity when mixture toxicity and background exposures are not taken into account.

A multi-generational study on low-dose BPA exposure in Wistar rats: Effects on maternal behavior, flavor intake and development

Bisphenol A (BPA) is a common endocrine disruptor found as an environmental and food contaminant. It exerts both developmental and behavioral effects, mainly when exposure occurs in early life. The aim of this study was to determine the multi-generational effects of chronic, human-relevant low-dose exposure to BPA on development, maternal behavior and flavor preference in Wistar rats.BPA was orally administered at a daily dose of 5μg/kg body weight to F0 pregnant dams from the first day of gestation (GD 1) until the last day of lactation (LD 21), and then to F1 offspring from weaning (PND 21) to adulthood (PND 100). F2 offspring were not exposed. Development and clinical signs of toxicity were assessed daily. Maternal behavior was evaluated by observing nursing and pup-caring actions, as well as “non-maternal” behaviors in F0 and F1 dams from parturition until LD 8. The flavor preferences of F1 and F2 offspring were evaluated based on the intake of sweet, salt and fat solutions using the two-bottle choice test on PND 21–34 and PND 86–99.BPA exposure: 1) decreased maternal behavior in F1 dams, 2) caused developmental defects in both F1 and F2 offspring, with a noticeable decrease in anogenital distance in male rats, and 3) did not affect flavored solution intake in F1, but induced changes in sweet preference in F2 juveniles and in salt and fat solution intakes in F2 adults, and 4) induced a body weight increase in the F2 generation only, whereas food intake and water consumption did not change.Taken as a whole, our findings showed that both gestational (F0) and lifelong (F1) exposures to a human-relevant dose of BPA could induce multi-generational effects on both development and behavior. These results suggest possible selective neuroendocrine defects and/or epigenetic changes caused by BPA exposure.

Adverse effect of tetracycline and doxycycline on testicular tissue and sperm parameters in CD1 outbred mice

Tetracycline and doxycycline are commonly used antibiotics in acne treatment during puberty in humans. The long-term effect of these antibiotics on male reproductive tract development has not been fully elucidated. For this reason we tested the effect of antibiotics on the reproductive parameters of mice males during puberty with the therapeutic dose used in humans, and with lower and higher doses. The outbred mouse strain CD1 with higher heterozygosity was exposed for 14 days at puberty. Adult males at the age of 70 days were used for the measurements. We observed a significant decrease in anogenital distance and thickness of the seminiferous epithelium in the treated animals. Pathological changes in the testes had an impact on sperm quality; a higher number of sperm positively stained with Annexin V and TUNEL and a lower number of acrosome-intact sperm was detected. In conclusion, the treatment of male mice with antibiotics in puberty led to long-lasting effects on reproductive organs and spermatozoa in adult males.

Fetal Growth Restriction Leads to Inhibition of Steroidogenesis.

Masculinization of the male reproductive tract occurs in humans from gestational weeks 10-22, and from gestational days 16-21 in rats. During this programming window, fetal testes produce a surge of testosterone causing reproductive masculinization. Disruption of this fetal testosterone production has been shown to result in various reproductive abnormalities including cryptorchidism and hypospadias. It is unknown what specifically causes these reproductive abnormalities aside from genetic defects of the steroidogenic pathway. In humans, in utero growth restriction is a well-known risk factor for cryptorchidism and hypospadias, but the mechanism is unknown. In addition, previous rat studies have suggested that fetal testosterone production can be disrupted by several factors including environmental exposure to anti-androgens such as dibutyl phthalate (DBP). In order to identify the mechanism underlying male reproductive malformation associated with in utero growth restriction, a maternal under-nutrition model was examined in the rat. Timed-pregnant Wistar rats were restricted to 50% (n=9) of the food eaten by ad libitum control dams (n=8) beginning on gestational day (GD) 3. Caloric restriction through GD17 resulted in a significant decrease in pup weight despite no significant decrease in litter size. Cultured fetal testes revealed a decrease in testosterone production of about 40% as indicated by radioimmunoassay. Additionally, mRNA levels of key components in the steroidogenic pathway including Scarb1 and Star were significantly decreased in the testis following in utero growth restriction. Analysis of postnatal endpoints following 50% caloric restriction (n=11) from GD3-18 indicated no evidence of undescended testes, hypospadias, and no significant decrease in anogenital distance at postnatal days 1, 14, or 28 as compared to ad libitum control animals (n=10). A coexposure study of in utero growth restriction and maternal dosing with the anti-androgen dibutyl phthalate was performed to determine if there was a cooperative effect on steroidogenesis. Analysis of fetal testes (GD17) following 50% maternal caloric restriction from GD3-17 (n=11) or exposure to DBP (250mg/kg bw/day) from GD15-17 without food restriction (n=7) showed a significant decrease in the expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star as compared to ad libitum controls (n=7). Moreover, when 50% caloric restriction was coupled with exposure to DBP (n=10), steroidogenic gene expression was further decreased. These results indicate that not only do in utero growth restriction and fetal exposure to DBP lead to a decrease in testosterone production separately, they also appear to work cooperatively to further disrupt the steroidogenic pathway. This interaction between chemical endocrine disruptor exposure and in utero growth restriction could begin to explain the mechanisms associated with human male reproductive abnormalities.

EHP Paper of the Year, 2009

The Paper of the Year Award was established in 2008 by Environmental Health Perspectives (EHP) as a means of reinforcing high-quality articles published in the journal, identifying emerging research themes, and tracking the impact of groundbreaking research (Tilson 2008). In this issue, we are pleased to announce that the EHP Paper of the Year for 2009 is “Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure” by Shanna H. Swan, Katharina M. Main, Fan Liu, Sara L. Stewart, Robin L. Kruse, Antonia M. Calafat, Catherine S. Mao, J. Bruce Redmon, Christine L. Ternand, Shannon Sullivan, J. Lynn Teague, and the Study for Future Families Research Team (Swan et al. 2005). We chose this paper because of its high impact in both the research and legislative realms since its publication in 2005. This paper (Swan et al. 2005) was the first to demonstrate an association between pregnant women’s exposure to phthalates and adverse effects on genital development in their male children. Rodent studies had previously identified a syndrome of adverse effects of phthalates on the male reproductive system (Foster 2006; Sharpe 2005), and find-ings from Swan et al. (2005) supported the hypothesis that prenatal phthalate exposure at environmental levels can also adversely affect male reproductive development in humans. These findings are important because humans are commonly exposed to phthalates found in a wide variety of consumer products, including soft vinyl items, medical tubing and IV bags, and a variety of personal care products such as perfume, lotion, shampoo, cosmetics, nail polish, and hairspray. Toxicologists routinely measure the external genitalia to assess reproductive toxicity in animal studies. One of these measures, anogenital distance (AGD)—a particularly sensitive indicator of masculinization—is shortened in male rodents following prenatal exposure to several phthalates (Foster 2006; Sharpe 2005). Swan et al. (2005) translated the standard animal exam to humans in order to investigate potential effects of phthalates on reproductive development in male infants. Specifically, they estimated associations between the presence and quantity of nine phthalate metabolites in mothers’ prenatal urine samples and AGD and other measurements in their sons. Higher levels of four phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl phthalate (MiBP)] were associated with a shorter AGD. Swan subsequently replicated and extended these findings (2008). Swan et al. (2005) has had an impact on phthalate legislation such as the Consumer Product Safety Improvement Act of 2008, which dramatically reduced the amount of six phthalates (including DEHP and DBP) that are permissible in children’s toys. The findings of Swan et al. (2005) have also been discussed and documented in numerous congressional hearings, including the June 2008 House Subcommittee on Commerce, Trade, and Consumer Protection hearing (Committee on Energy and Commerce 2008). This research was also cited heavily in support of regulations passed in California, Vermont, and Washington and introduced in Maine, New York, New Jersey, Connecticut, Maryland, Massachusetts, Rhode Island, West Virginia, Minnesota, Illinois, Oregon, and Hawaii. The AGD measure developed by Swan et al. (2005) is now being incorporated into multiple ongoing studies. The National Children’s Study (NCS), for example, proposes to include AGD using a similar protocol in the infant exam being piloted in the NCS Vanguard Centers (National Children’s Study 2009). In addition, Swan and colleagues expect funding in mid-2009 for a new multi center pregnancy cohort study that will examine prenatal phthalate exposure in relation to AGD and other reproductive parameters in a larger population using more precise methods. This research will provide standards for measuring genital landmarks obtained in a diverse population-based sample of male infants, which may be suitable for use in pediatric practice. Associations between phthalate metabolites and these androgen-sensitive measures could further impact public health policy, given the ubiquitous nature of these exposures and the clinical importance of appropriate androgen stimulation during fetal development.

EHP Papers of the Year, 2008

<i>EHP</i> Papers of the Year, 2008

The Conquest of Lead Poisoning: A Pyrrhic Victory

The Conquest of Lead Poisoning: A Pyrrhic Victory

Anogenital Distance: Defining “Normal”

In their letter to EHP, McEwen and Renner (2006) dismissed the findings of Swan et al. (2005), who reported a significant relationship between a measure of anogenital distance (AGD) in boys and levels of phthalate metabolites in their mothers’ urine during pregnancy. AGD is a sexually dimorphic index that, on average, is twice as great in males as in females, so it serves as a marker of proper male development. McEwen and Renner based their argument on an idiosyncratic form of logic. They asserted that All male infants evaluated in the study appeared normal … there is no evidence for potential adverse effect in the test population. … no conclusion can be drawn whether the reported values are normal or abnormal. The range of AGD values … likely represents typical biologic variation that would be expected to occur among normal study subjects. McEwen and Renner seem to be wholly unfamiliar with the meaning of a modest or even a slight shift in the mean of an index that reflects the distribution of susceptibility in a population. I have pointed out (Weiss 1988) that even a 5-point (5%) reduction in mean intelligence quotient in a population of 100 million increases the number of individuals classified as retarded from 6 million to 9.4 million. It is this kind of relationship that eventually prompted the Centers for Disease Control and Prevention (CDC) to lower its definition of elevated lead risk levels in blood, set at 40 μg/dL in 1970, to 10 μg/dL in 1991 (CDC 1991). Bellinger (2006) put it this way: A small change in the mean signals predictable accompanying changes in the proportions of individuals in the source population who fall into the tails of the distribution, where individuals who meet diagnostic criteria are found. Thus, the importance of a shift in group mean lies not in what it indicates about the average change among members of the study sample, but what it implies about the changes in the tails of the distribution in the population from which the study sample was drawn. He noted, based on Rose (1981), that in a population with a prevalence of clinically defined hypertension of 15%, a 5-mm reduction in mean systolic blood pressure would result in a 33% decrease in prevalence (Bellinger 2006). Epidemiologists recognize that a slight decrease in mean blood pressure in a population is translated into a major decrease in the incidence of serious cardiovascular events such as heart attacks. We already know that shortened AGD at birth is one element, the leading edge, as it were, of the “phthalate syndrome” in rats, which is marked by testicular pathology, reduced spermatogenesis, hypospadias, and cryptorchidism, a compilation of signs indicating disordered male development that Sharpe (2001) and others have noted to be on the increase in industrialized nations. An almost imperceptible shift to a lower mean AGD in the human male would foreshadow a heightened prevalence of reproductive system dysfunction. Is that the connection now emerging in the clinic? If McEwen and Renner’s (2006) criteria for “normal” were to govern the way in which we define the health risks of lead exposure, we would be basing our criteria on the number of children brought into hospital emergency rooms with lead poisoning rather than on the threats it poses to their neurobehavioral development. No parent, and no community, would tolerate such a definition these days.

Prenatal Phthalate Exposure and Anogenital Distance in Male Infants

In our article “Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure” (Swan et al. 2005), we reported results of our recent study on the relationship between anogenital distance (AGD) in boys and their mother’s urinary concentration of phthalate metabolites (Swan et al. 2005). The primary question we addressed was the relationship between the concentration of phthalate metabolites in maternal prenatal urine and the AGD, or the more appropriate derived measure anogenital index (AGI = AGD/weight), in human male offspring. We designed our study to focus on this specific measurement because of highly reliable results in the animal literature showing that certain phthalates reduce AGD (and AGI) in rodents and because, as continuous variables, AGD and AGI would not require a large sample size to demonstrate this relationship, if it existed. Changes in the frequency of a dichotomous and relatively rare end point such as frank cryptorchidism, also caused in animals by prenatal phthalate exposure, require far larger sample sizes. Secondarily, we looked at AGI in relation to other genital measurements (penile volume, testicular descent, and scrotal size), examining these interrelationships in several ways. In our article (Swan et al. 2005), we reported that urinary concentrations of four phthalate metabolites [mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), monoethyl phthalate (MEP), and mono-isobutyl phthalate (MiBP)] were inversely and significantly related to AGI. We also examined three metabolites of diethylhexyl phthalate (DEHP). Although the associations between AGD and the secondary DEHP metabolites [mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP)] were suggestive, they were not statistically significant, and the metabolite MEHP appeared to be unrelated to AGI. We examined the relationship between AGI and testicular descent in several ways, varying whether each of these variables was entered into the analysis untransformed (e.g., as they were recorded in the examination) or as dichotomous variables. AGD was measured by the examiner using a Vernier calipers. Both AGD and AGI are continuous, and approximately normally distributed, variables. The degree of descent of each testicle was categorized as follows: 0 = normal, 1 = normal retractile, 2 = high scrotal, 3 = suprascrotal, 4 = inguinal, and 5 = nonpalpable or ectopic. The testicular placement score (TPS) is the sum of the recorded value for the left and right testicle. Therefore, the lower the TPS, the more complete the testicular descent. We first examined the relationship between AGI and testicular descent by calculating the correlation coefficient between AGI and TPS (an ordinal variable). In the complete data set, including 134 boys with genital examination, AGI is significantly and inversely related to TPS (correlation coefficient −0.201, p-value 0.021). That is, shorter AGI was significantly associated with less complete testicular descent. This analysis assumes that TPS is an interval variable; for example, the difference between a score of 0 (both testicles “normal”) and 1 (one testicle “normal” and one “normal retractile”) is equal to that between 1 and 2 (either both “normal retractile,” or one “normal” and one “high scrotal”). We also examined TPS as a dichotomous variable, which does not require this assumption. For this purpose, testicular descent was coded as 0 and called “complete” if both testicles were rated as either normal or normal retractile; otherwise, it was coded as 1 and called “incomplete.” This dichotomous variable was also significantly correlated with AGI (correlation coefficient −0.192, p-value 0.027). Since results by these two methods were similar, we did not include this latter analysis in our article (Swan et al. 2005). As is common practice in epidemiologic analyses, we also dichotomized AGI to create two groups to serve as cases and controls. For this purpose, we classified boys into “short” AGI (< 25% of expected for age) or “not short.” We looked at the proportion of boys with incomplete testicular descent in these groups. We incorrectly stated the p-value for not short (0.136) in our article (Swan et al. 2005) to be statistically significant. That this analysis was not statistically significant, while the analysis of the AGI as continuous variable was, is not surprising; dichotomizing a continuous variable results in a loss of power, and thus a larger sample size is needed to achieve a similar level of statistical significance (Ragland 2002). In conclusion, in all analyses boys with shorter AGI had less complete testicular descent, and significantly so for the two analyses in which AGI was treated as a continuous variable. The miscalculation of statistical significance for one analysis, while unfortunate, in no way alters any of our conclusions. Also, this error does not weaken this article’s (Swan et al. 2005) support for the importance of examining patterns of subtle changes in humans, as suggested by toxicology, when assessing the effects of environmental exposures.

Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure

Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in male rodents. We present data from the first study to examine AGD and other genital measurements in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was obtained in 134 boys 2–36 months of age. AGD was significantly correlated with penile volume (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20, p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors of age-adjusted AGI in regression and categorical analyses that included all participants with prenatal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP concentration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age-adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009). The associations between male genital development and phthalate exposure seen here are consistent with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally exposed rodents. The median concentrations of phthalate metabolites that are associated with short AGI and incomplete testicular descent are below those found in one-quarter of the female population of the United States, based on a nationwide sample. These data support the hypothesis that prenatal phthalate exposure at environmental levels can adversely affect male reproductive development in humans.

Phthalate Exposure and Early Thelarche

Phthalate Exposure and Early Thelarche

Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat.

Finasteride is a specific inhibitor of type II 5alpha-reductase, the enzyme that converts testosterone (T) to the more potent androgen receptor agonist dihydrotestosterone (DHT). In utero exposure to androgen receptor antagonists and T biosynthesis inhibitors have induced permanent effects on androgen-sensitive end points such as anogenital distance (AGD), nipple retention, and malformations of the male rat reproductive tract. The objectives of this study were to (1) characterize the dose response of finasteride-mediated alterations in androgen-dependent developmental end points, (2) determine whether prenatal exposure to finasteride permanently decreases AGD or results in nipple retention, and (3) evaluate whether AGD or nipple retention is predictive of adverse alterations in the male reproductive tract. Pregnant Crl:CD(SD)BR rats (n=5-6/group) were gavaged with either vehicle or finasteride at 0.01, 0.1, 1.0, 10, or 100 mg/kg/day on gestation days 12 to 21. All male offspring were monitored individually until necropsy on postnatal day (PND) 90. The present study design has been used previously for other antiandrogens and is sensitive to perturbations of the male rat reproductive tract. Decreases in AGD on PND 1 and increases in areolae-nipple retention on PND 13 were significantly different from controls in all finasteride-exposed male rats. Finasteride-induced changes in AGD and nipple retention were permanent in male rats exposed to finasteride at and above 0.1 mg/kg/day. On PND 90, dorsolateral and ventral prostate lobes were absent in 21 to 24% of rats exposed to 100 mg/kg/day finasteride and weighed significantly less at and above 10 mg/kg/day. In the highest dose group, 73% of animals had ectopic testes, much higher than previously reported. The most sensitive malformation other than decreased AGD and nipple retention was the dose-dependent increase in hypospadias. The lowest observed adverse effect level (LOAEL) for finasteride-induced permanent effects in this study was 0.1 mg/kg/day based on permanent changes in AGD and nipple retention. Finasteride-induced changes in AGD and retention of nipples were highly predictive of hypospadias, ectopic testes, and prostate malformations even though some animals with retained nipples or decreased AGD may not have had other reproductive tract malformations. In summary, prenatal exposure to finasteride specifically inhibited DHT-mediated development with little to no change in T-mediated development.

Comparison of antiandrogenic activities of vinclozolin and d,l-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo

A chemical substance used as a photoinitiator for light-cure resin compositions, d,l-camphorquinone (CQN) was found to be weakly antiandrogenic in vitro. It competitively antagonized dihydrotestosterone (DHT)-induced transcriptional activity on the yeast-based androgen receptor gene transcription assay (YAA). Antiandrogenic activity of CQN was shown at higher concentration than 10 −4 M while the well-known antiandrogen, vinclozolin (VCZ) showed the activity at concentrations of 10 −6 M and above in YAA. The antiandrogenic activity of CQN was reconfirmed in the human cell line-based androgen receptor gene transcription assay (HCAA). To determine whether CQN affect male reproductive development, CQN or VCZ was administered to pregnant mice daily from gestational days 10 to 18 by gavage. In utero exposure to VCZ at 100 mg/kg/day caused a significant decrease in anogenital distance (AGD) of F1 neonates and reduced spermatogenesis in F1 males at 42 days of age. In contrast, maternal doses (100 and 300 mg/kg/day) of CQN had no affect on these endpoints in F1 offspring. Further, VCZ or CQN had no adverse affect on F1 male fertility. From these observations, CQN is potentially antagonistic to androgen receptor (AR) in vitro, but is estimated to be less antiandrogenic in vivo when it is administered to pregnant mice by gavage. Furthermore, these findings are the first to demonstrate that VCZ exerts significant antiandrogenic effects on reproductive tract development during gestation in mice.

Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy.

Prenatal exposure to the herbicide linuron, a weak androgen receptor antagonist, has been shown to perturb androgen-dependent male rat reproductive development as evidenced by slight decreases in anogenital distance (AGD), increased retention of areolae/nipples, and induction of epididymal malformations in combination with testicular atrophy in the adult rat over dose levels ranging from 12.5 to 100 mg/kg/day. Studies were undertaken to determine whether linuron-mediated changes in AGD and nipple retention are permanent, whether linuron is a direct testicular toxicant, and if there was an association between areola/nipple retention and malformations. Pregnant rats were administered corn oil vehicle or linuron by gavage at 0 or 50 mg/kg/day (n = 8 controls, 20 treated) from gestation days 12 to 21. Male offspring were necropsied on postnatal days (PND) 35 and 56. Linuron-exposed male rats exhibited a significant (8%) decrease in AGD on PND 1 and a similar decrease was also observed on PND 56. Linuron-exposed male rats displayed an increase in areola retention on PND 13, as evidenced by 0.6 +/- 0.5 and 3.3 +/- 0.4 areolae per rat in the control and exposed groups, respectively. Male rats displayed a significant increase in nipple retention on PND 35 and 56 (collectively) of 0 +/- 0.5 and 1.7 +/- 0.3 nipples per rat in control and exposed groups, respectively. On PND 35, 4/51 rats (3/9 litters) from linuron-treated dams displayed enlarged testes in combination with malformed epididymides. Epididymal malformations were observed in 19/51 rats (6/9 litters) in the linuron-exposed dose group. On PND 56, grossly enlarged and edematous testes were seen in 16/56 linuron-exposed rats (6/9 litters). Epididymal lesions were observed in 23/58 rats (6/9 litters). Microscopically, all linuron-exposed animals that exhibited a testicular lesion on PND 56 also displayed an epididymal lesion. These lesions were not seen in control animals. Approximately 25 and 60% of the male offspring that had malformations of the epididymis and vas deferens did not exhibit either areolae on PND 13 or nipples at necropsy, respectively. These data indicate that in utero linuron exposure to 50 mg/kg/day results in permanent changes in AGD and nipple retention in male rats. Moreover, these findings indicate that linuron-induced testicular atrophy, which is observed in adult rats, is secondary to increased intratubular pressure resulting from obstruction of testicular fluid outflow subsequent to malformation of the epididymides. These data also suggest that although linuron-mediated retention of areolae on PND 13 and nipples at necropsy may be suggestive of altered testosterone-mediated reproductive development seen in adult rats, these endpoints are not predictive.

Effect of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126) throughout gestation and lactation on development and spatial delayed alternation performance in rats.

There is evidence that polychlorinated biphenyl (PCB) congeners have differential effects on endpoints of neurotoxicity depending on their chemical structure: specifically, that ortho-substituted congeners are neurotoxic whereas coplanar (dioxin-like) congeners are relatively inactive in producing neurotoxic effects. The effects of the coplanar congener 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on developmental endpoints, hematology, serum biochemistry, and performance on a spatial delayed alternation task were assessed in Long-Evans rats. Dams were dosed with 0, 0.25, or 1.0 microg/kg/day Monday to Friday beginning 5 weeks before and continuing through gestation and lactation. The first 2-week breeding period produced 10, 8, and 13 litters in the three dose groups, respectively. Breeding females from the control and low-dose group that did not conceive were rebred after 76 days of dosing, producing 7 and 6 litters, respectively. Reduction in weight gain from birth to weaning at 21 days of age (DOA) was observed in both dose groups of Cohort 1 but not in Cohort 2. Males in Cohort 1 exhibited a slight decrease in anogenital distance normalized for weight. Changes in hematological and some serum biochemical parameters were observed in the pups at DOA 21 and/or 60. PCB 126 was detected in fat sampled at both DOA 21 and 60. PCB 126 was not detected in brain samples at 60 DOA in any group; analysis of Cohort 2 at DOA 21 revealed levels in the treated group about 1/100 of those in fat. On the spatial delayed alternation task, there was no convincing evidence for impairment as a result of PCB exposure, as assessed by overall accuracy of performance and measures of perseverative and other types of inappropriate responding. These data provide further evidence for the lack of neurotoxicity of dioxin-like PCB congeners. However, assessment of performance on additional behavioral indices is required before definitive conclusions may be drawn.

Critical Developmental Periods for Effects on Male Rat Genitalia Induced by Finasteride, a 5α-Reductase Inhibitor

The conversion of testosterone to 5α-dihydrotestosterone by the enzyme 5α-reductase is inhibited by finasteride. In a study in which maternal dosing with finasteride commenced on Gestational Day 15 and terminated on Postpartum Day 21, there were 13 and 27% decreases in anogenital distance of male pups on Postnatal Day 1 at 0.03 and 3 mg/kg/day, respectively. These decreases were largely reversed by Postnatal Day 22 even though treatment of the dams continued. Treatment at 3 mg/kg/day also resulted in hypospadias with cleft prepuce and a 5-day delay in the separation of the prepuce from the glans penis in those animals without hypospadias. A second study in which 20 mg/kg/day finasteride was administered on successive 2-day periods during late gestation in rats demonstrated that the period of Gestational Days 16 to 17 was the most sensitive (critical period) for finasteride-induced hypospadias, cleft prepuce, decreased anogenital distance, reduced prostate weight, and nipple formation in F1 male offspring. This critical period is just prior to the appearance on Day 18 of gestation of a midline mesenchymal plate between the urogenital sinus and the rectum in normal male fetuses. This midline plate does not appear in finasteride-exposed fetuses destined to have hypospadias as demonstrated in a previous study. Based on these observations. we hypothesize that finasteride causes hypospadias by preventing the formation of the medial mesenchymal plate which is necessary for assisting the movement of the urogenital sinus from the base to the tip of the genital tubercle.