Applying the adverse outcome pathway concept for assessing non-monotonic dose responses: biphasic effect of bis(2-ethylhexyl) phthalate (DEHP) on testosterone levels.

Male reproduction is one of the primary health endpoints identified in rodent studies for some phthalates, such as DEHP (Bis(2-ethylhexyl) phthalate), DBP (Dibutyl phthalate), and BBP (Benzyl butyl phthalate). The reduction in testosterone level was used as an intermediate key event for grouping some phthalates and to establish a reference point for risk assessment. Phthalates, and specifically DEHP, are one of the chemicals for which the greatest number of non-monotonic dose responses (NMDRs) are observed. These NMDRs cover different endpoints and situations, often including testosterone levels. The presence of NMDR has been the subject of some debate within the area of chemical risk assessment, which is traditionally anchored around driving health-based guidance values for apical endpoints that typically follow a clear monotonic dose-response. The consequence of NMDR for chemical risk assessment has recently received considerable attention amongst regulatory agencies, which confirmed its relevance particularly for receptor-mediated effects. The present review explores the relationship between DEHP exposure and testosterone levels, investigating the biological plausibility of the observed NMDRs. The Adverse Outcome Pathway (AOP) concept is applied to integrate NMDRs into Key Event Relationships (KERs) for exploring a mechanistic understanding of initial key events and possibly associated reproductive and non-reproductive adverse outcomes.

Indoor PM2.5, VOCs and asthma outcomes: A systematic review in adults and their home environments.

INTRODUCTION: As the amount of time people spend indoors increases globally, exposure to indoor air pollutants has become an important public health concern. Asthma is a complex disease caused and/or exacerbated by increased exposure to diverse chemical, physical and biological exposures from multiple indoor and outdoor sources. This review aims to investigate the relationship between increased indoor PM and VOC concentrations (i.e. objectively measured) and the risk of adult asthma in higher-income countries. METHODS: Eleven databases were systematically searched on the February 1, 2019 and again on the February 2, 2020. Articles were limited to those published since 1990. Reference lists were independently screened by three reviewers and authors were contacted to identify relevant articles. Backwards and forward citation chasing was used to identify further studies. Data were extracted from included studies meeting our eligibility criteria by three reviewers and assessed for quality using the Newcastle-Ottawa scale designed for case-control and cohort studies. RESULTS: Twelve studies were included in a narrative synthesis. We found insufficient evidence to determine the effect of PM2.5 on asthma in the indoor home environment. However, there was strong evidence to suggest that VOCs, especially aromatic compounds, and aliphatic compounds, were associated with increased asthma symptoms. DISCUSSION & CONCLUSION: Although no single exposure appears to be responsible for the development of asthma or its associated symptoms, the use of everyday products may be associated with increased asthma symptoms. To prevent poor health outcomes among the general population, health professionals and industry must make a concerted effort to better inform the general population of the importance of appropriate use of and storage of chemicals within the home as well as better health messaging on product labelling.

Lessons from a publicly funded tier 2 healthy weight programme in Cornwall, UK.

BACKGROUND: The UK has one of the highest prevalence rates of obesity worldwide. Public health departments have a duty to provide some obesity treatment and prevention services. With evidence of effective programmes lacking, we investigate lessons learned from a healthy weight programme in Cornwall, UK. METHODS: Data from the 12-week multi-component adult healthy weight management programme were obtained for 2012-2016. Descriptive statistics and statistical tests were used to describe participants' demographics, health status and anthropometric measures to explore the enrolment and retention of the programme as well as the impact. RESULTS: A total of 1872 adults were referred into the programme. Overall, 646 completed the programme and, 48.8% achieved the programme's aim of a >3% reduction in weight. Those who completed and met the programme aim tended to have had healthier outcomes at baseline. CONCLUSIONS: For those who engage with the programme the impact can be meaningful. However, <1% of the population of Cornwall with overweight or obesity enroled in the programme, and those who benefitted most might have been in least need. Providing services that meet the needs of the population is challenging when a variety of services is needed, and the evidence base is poor.

Variable risk of atopic disease due to indoor fungal exposure in NHANES 2005-2006.

BACKGROUND: Exposure to damp indoor environments is associated with increased risk of eczema, allergy and asthma. The role of dampness-related exposures and risk of allergic diseases are yet to be fully explored in the US population. OBJECTIVE: We assess whether exposure to fungi, house dust mites and endotoxin increases the risk of eczema, allergy and asthma in children and adults participating in NHANES 2005-2006. METHODS: A total of 8412 participants (2849 were children aged between 6 and 17 years) were recruited in the 2005-2006 survey. We used multiple logistic regression to investigate whether mildew/musty odour and increased concentrations of Alternaria alternata allergen, Aspergillus fumigatus antigens, house dust mite and endotoxin antigens increase the risk of eczema, allergy and asthma. We stratified models by total IgE < 170 and ≥ 170 KU/L to assess allergic and non-allergic asthma outcomes. Exposure to multiple biological agents and risk of reporting eczema, allergy and asthma were also investigated. RESULTS: Reporting of a mildew/musty odour was associated with increased risk of childhood asthma (OR 1.60; 95% CI 1.17-2.19), and adult eczema, allergy and asthma (OR 1.92; 95% CI 1.39-2.63, OR 1.59 95% CI 1.26-2.02 and OR 1.61 95% CI 1.00-2.57, respectively). Risk of asthma was associated with total IgE ≥ 170 KU/L in children (OR 1.81; 95% CI 1.01-3.25) and total IgE < 170 KU/L in adults (OR 1.91; 95% CI 1.07-3.42). Children and adults exposed to more than eight biological agents present in the home were at reduced risk of eczema (OR 0.17; 95% CI 0.04-0.77) and asthma (OR 0.49; 95% CI 0.25-0.97), respectively. CONCLUSION: Exposure to a mildew/musty odour, as a proxy for exposure to fungus, was implicated in an increased risk of atopic diseases. Sensitisation may play a different role in children and adults, and exposure to multiple allergens may reduce the risk of atopic disease.

Description of Ophthalmic Pharmaceutical and Device Start-Up Companies.

AIMS: To describe the number, type and location of ophthalmic companies and their associated product areas and indications. METHODS: A retrospective, non-patient-based, observational review of ophthalmic pharmaceutical and device companies with a new product in development. Data was compiled by Internet searches. RESULTS: We identified 190 companies currently developing ophthalmic products: 134 (71%) were privately held and 56 (29%) publicly held, while 136 (72%) were in the United States and 53 (28%) were outside the United States. There were 436 total products of which 338 (78%) were pharmaceuticals and 98 (22%) devices. With pharmaceuticals we identified 46 separate indications with age-related macular degeneration (n = 75), glaucoma (n = 52) and dry eye (n = 46) as most common; anti-vascular endothelial growth factor, hormone therapy and anti-inflammatory products were also common classes. With devices there were 30 indications with glaucoma (n = 26), age-related macular degeneration (n = 19) and dry eye (n = 6) as most common; drug delivery, ocular implants and prostheses were less common classes. CONCLUSIONS: Ophthalmology as a specialty is benefited by a wide effort in new medicine and device development. However, a concentration of effort into relatively few indications suggests a potential lack of market analysis and possible difficulty for many companies in commercializing their product.

Techniques to Reduce the Placebo Effect in Glaucoma Clinical Trials.

AIM: To evaluate techniques used to reduce the placebo effect in prior well-controlled, single or double-masked placebo-controlled glaucoma trials. METHODS: This study was a retrospective, non-patient-based, observational review of phase I-III trials with a placebo arm for glaucoma medicines available after 1977. RESULTS: This study included 20 articles with 20 placebo control arms consisting of 458 patients evaluating 10 different glaucoma medications with 58 treatment arms. There was no statistical difference across the evaluated types of study designs to limit the placebo effect either for the morning trough or diurnal curve. The average reduction of the intraocular pressure in the placebo groups was 1.6 ± 1.5 mm Hg for the morning trough and 1.3 ± 1.3 mm Hg for the diurnal curve across all studies. CONCLUSION: The results of this study suggest that current design techniques described in the literature to limit the placebo effect appear ineffective compared to no additional techniques.

Modifiable factors governing indoor fungal diversity and risk of asthma.

Exposure to dampness and fungi in the home is a known risk factor for individuals with allergic asthma. Inadequate heating and ventilation may lead to dampness and concomitant increased exposure to spores of allergenic fungi such as Aspergillus and Penicillium. These fungi have been cultured from sputum of asthmatic and non-asthmatic individuals, and implicated in the initiation or exacerbation of asthma. Indoor environmental factors influence the presence and concentrations of fungal propagules and, in turn, risk of asthma outcomes. This review aims to identify modifiable risk factors in the built environment that have been shown to influence fungal composition indoors, and to examine this association with the risk of asthma development and/or exacerbation. A complex interaction between residential characteristics, the built environment and the behaviour of people regulate the diversity and concentrations of indoor fungi. Modifiable factors include build age, architectural design, level of maintenance, variations in construction materials, presence of pets, heating and ventilation patterns. Risk of fungal contamination and asthma outcomes are also influenced by low occupant awareness concerning potential health effects and socio-economic factors. Addressing these factors provides an opportunity to improve future housing interventions, though it is not clear how the built environment and occupant behaviours interact to modify the diversity of indoor fungi and resultant risk of asthma. A combination of housing improvements combined with awareness programmes and the alleviation of fuel poverty can be used to lower the allergen burden associated with damp homes. Further research is needed to identify factors that regulate the concentration and diversity of indoor fungi and how this may act as a modifier for asthma outcomes.

Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration.

AIMS: To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). METHODS AND RESULTS: Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. CONCLUSIONS: Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration.

Changes in middle cerebral artery velocity after carotid endarterectomy do not identify patients at high-risk of suffering intracranial haemorrhage or stroke due to hyperperfusion syndrome.

OBJECTIVES: To determine if significant increases in middle cerebral artery velocity (MCAV) or pulsatility index (PI) during and immediately after carotid endarterectomy (CEA) were predictive of patients suffering a stroke due to the hyperperfusion syndrome (HS) or intracerebral haemorrhage (ICH). METHODS: Transcranial Doppler (TCD) mean/peak MCAV and PI were recorded pre-operatively; pre-clamp; 1-min post-declamping; 10-min post-declamping and 30-min post-operatively. The study was divided into two time periods; Group 1 (1995-2007); where there was no formal guidance for managing post-CEA hypertension (PEH) and Group 2 (2008-2012); where written guidelines for treating PEH were available. RESULTS: 11/1024 patients in Group 1 (1.1%) suffered a stroke due to HS/ICH, compared to 0/426 patients (0.0%) in Group 2 (p = 0.02). In Group 1; intra-operative increases >100% in mean/peak MCAV and PI at 1 and 10-min post-clamp release had positive predictive values (PPV) of 1.2%, 6.3% and 20.0% and 2.9%, 8.0% and 16.6% respectively. Post-operatively; a >100% increase in mean and peak MCAV had a PPV of 6.3% and 2.7% respectively. CONCLUSION: We were unable to demonstrate that significant increases in MCAV and PI were able to predict patients at increased risk of suffering a post-operative stroke due to HS or ICH. The provision of written guidance for managing PEH in Group 2 patients was associated with virtual abolition of ICH/HS.

Procedural risk following carotid endarterectomy in the hyperacute period after onset of symptoms.

OBJECTIVES: There have been concerns that performing carotid endarterectomy (CEA) in the hyperacute period after onset of a transient ischaemic attack (TIA) or stroke may be associated with a significant increase in the procedural risk that could offset any long-term benefit to the patient. The aim of this audit was to determine the 30-day risk of stroke/death after CEA in symptomatic patients, stratified for delay from the most recent neurological event, mode of presentation, and age. METHODS: Retrospective audit in 475 recently symptomatic patients between October 1, 2008, and April 24, 2013. RESULTS: Forty-one patients (9%) underwent surgery <48 hours of their most recent event, with a 30-day death/stroke rate of 2.4% (1/41). The procedural risk was 1.8% in 167 patients who underwent surgery within 3-7 days (3/167), falling to 0.8% in 133 patients who underwent surgery between 8 and 14 days (1/133) and 0.8% in 134 patients whose surgery took place after >14 days had elapsed (1/134). Overall, 208 (44%) underwent surgery within 7 days of their most recent neurological event (30-day risk = 1.9%), while 341 (72%) underwent CEA within 14 days (30 day risk = 1.5%). There was no evidence of any systematic differences in procedural risk by operating in the hyperacute period relating to mode of presentation (TIA, stroke, amaurosis) or age (<80 years; >80 years). CONCLUSIONS: This audit found no evidence that the procedural risk was increased when CEA was performed in the hyperacute period whether this time period was defined as <48 hours, <7 days, or <14 days.

The effect of dihydrotestosterone exposure during or prior to the masculinization programming window on reproductive development in male and female rats.

Masculinization is programmed by androgen exposure during a masculinization programming window (MPW). Deficiency in MPW androgen action results in reduced size of all reproductive organs and anogenital distance (AGD) and reproductive disorders. Although timing of MPW closing has been defined, what determines 'opening' and 'closing' of the MPW remains unknown. To test whether initiation of testosterone production/action defines the opening of the window, we first demonstrated that androgen receptor mRNA and protein are expressed prior to the MPW, and then investigated whether masculinization could be advanced or enhanced by treating pregnant rats with either 1 or 10 mg/kg/day dihydrotestosterone (DHT) prior to (early window, EW; e11.5-e14.5) or during the MPW (e15.5-e18.5), and then evaluating offspring in foetal life (e18.5, e21.5), early puberty (day 25) or adulthood (∼day 75). DHT treatment did not affect pregnancy duration, birth, litter or pup size. DHT exposure in either time window did not advance foetal male development (Wolffian duct coiling) and had no effect on AGD, testis, penis and ventral prostate (VP) size at any age when measured; there was a tendency towards smaller penis size. In contrast, exposure of females to 10 mg DHT in either time window induced varying degrees of masculinization, including stabilization of the Wolffian duct and increased AGD (e21.5, Pnd25), VP formation, more male-like phallus structure, absence of nipples and vaginal opening and, in some adult females, gross fluid distension of the uterus (hydrometrocolpos); these effects were generally more pronounced after exposure in the MPW than in the EW. In conclusion, exposure of the male rat foetus to additional androgens prior to or during the MPW does not advance or enhance any measured parameter of reproductive development. Therefore, androgen availability plays no role in determining timing of the MPW. Susceptibility of the female reproductive system to androgens may precede the MPW.

Effects of inducible nitric oxide synthase (iNOS) deficiency in mice on Sertoli cell proliferation and perinatal testis development.

Nitric oxide (NO) plays crucial roles in several physiological and pathological conditions. The iNOS isoform produces high levels of NO independent of intracellular calcium and, in the testis, which is expressed in Sertoli (SC), Leydig (LC) and germ cells. The testicular roles of NO are unclear, but it can inhibit LC testosterone production. Our aim was to evaluate the effects of iNOS deficiency on testis development in mice from late fetal life through early puberty. Therefore, testes from wild type (C57BCL/6) and iNOS(-/-) mice (B6.129P2- Nos2(tm1Lau) /J) were sampled at various ages between e18.5 and Pnd20 and evaluated by histological and stereological analyses; proliferating cells were labelled with (3)H-thymidine. At all ages, testis weight and anogenital index, a measure of fetal androgen exposure, were greater in iNOS-deficient mice than in wild type mice. At all ages after birth, iNOS-deficient mice exhibited increased (p < 0.05) SC number per testis, and this was accounted for by a higher SC proliferation index (p < 0.05) in iNOS-deficient mice, especially on Pnd1 and Pnd5. Similarly, LC number per testis was higher (p < 0.05) in iNOS(-/-) mice than in wild type at all post-natal ages. Highly positive and significant correlations were observed between the proliferation index for SC, LC and peritubular myoid cells on e18.5 and post-natally. Although lumen formation was slightly advanced in iNOS(-/-) mice, no obvious other effects on pubertal testis development were observed. These results imply that NO may normally constrain testis somatic cell development, especially SC, perhaps by limiting testosterone production. Removal of this constraint results in normal, but larger, testes with greater sperm production. Our data pinpoint the window of iNOS (NO) action on SC proliferation and raise the possibility that experimental manipulation of NO in early post-natal life could be used to enhance SC proliferation if this was deficient for any reason.

Androgen receptor signalling in peritubular myoid cells is essential for normal differentiation and function of adult Leydig cells.

Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number, but resulted in compensated LC failure. The current study extends these investigations, demonstrating that PTM AR signalling is important for normal development, ultrastructure and function of adult LCs. Notably, mRNAs for LC markers [e.g. steroidogenic factor 1 (Nr5a1), insulin-like growth factor (Igf-1) and insulin-like factor 3 (Insl3)] were significantly reduced in adult PTM-ARKOs, but not all LCs were similarly affected. Two LC sub-populations were identified, one apparently 'normal' sub-population that expressed adult LC markers and steroidogenic enzymes as in controls, and another 'abnormal' sub-population that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike 'normal' LCs in PTM-ARKOs, the 'abnormal' LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC sub-populations is likely to mean that the 'normal' LCs work harder to compensate for the 'abnormal' LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM-ARKOs.

Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals.

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

The war against error: a 15 year experience of completion angioscopy following carotid endarterectomy.

BACKGROUND: A policy of intra-operative transcranial Doppler (TCD) and completion angioscopy was previously associated with virtual abolition of intra-operative stroke (apparent upon recovery from anaesthesia) following carotid endarterectomy (CEA). The aims of this study were to determine whether the prevalence of technical error has diminished with experience and whether our monitoring/quality control policy was still associated with low rates of intra-operative stroke 20 years after its introduction. METHODS: Retrospective review of four consecutive cohorts of 400 patients undergoing CEA between October 1995 and March 2010 (1600 CEAS in total). RESULTS: One hundred four patients (7%) had thrombus removed following angioscopy and prior to flow restoration, while 31 (2.1%) underwent repair of a distal intimal flap. The prevalence of intimal flaps diminished from 4.9% in the first 400 patients to 0.8% in the last 400 patients (p = 0.006). By contrast, the prevalence of retained thrombus did not decline with experience (8.5%, 3.7%, 10.3% and 5.4% for the four consecutive periods). Intra-operative TCD and completion angioscopy was, however, associated with extremely low rates of intra-operative stroke (0.25%, 0.25%, 0.5% and 0.25% during the four study periods). CONCLUSION: Most intra-operative strokes probably follow embolisation of thrombus following restoration of flow. This can be prevented by angioscopy which has the advantage of being performed prior to flow restoration. Increasing experience was associated with a decline in the detection of intimal flaps, but not in the prevalence of retained thrombus. Even the most experienced of surgeons can still be responsible for inadvertent technical error.

Effects of di(n-butyl) phthalate exposure on foetal rat germ-cell number and differentiation: identification of age-specific windows of vulnerability.

Environmental factors are implicated in increased incidence of human testicular germ-cell cancer (TGCC). TGCC has foetal origins and may be one component of a testicular dysgenesis syndrome (TDS). Certain phthalates induce TDS in rats, including effects on foetal germ cells (GC). As humans are widely exposed to phthalates, study of the effects of phthalates on foetal rat GC could provide an insight into the vulnerability of foetal GC to disruption by environmental factors, and thus to origins of TGCC. This study has therefore characterized foetal GC development in rats after in utero exposure to di(n-butyl) phthalate (DBP) with emphasis on GC numbers/proliferation, differentiation and time course for inducing effects. Pregnant rats were treated orally from embryonic day 13.5 (e13.5) with 500 mg/kg/day DBP for varying periods. GC number, proliferation, apoptosis, differentiation (loss of OCT4, DMRT1 expression, DMRT1 re-expression, GC migration) and aggregation were evaluated at various foetal and postnatal ages. DBP exposure reduced foetal GC number by ∼60% by e15.5 and prolonged GC proliferation, OCT4 and DMRT1 immunoexpression; these effects were induced in the period immediately after testis differentiation (e13.5-e15.5). In contrast, DBP-induced GC aggregation stemmed from late gestation effects (beyond e19.5). Foetal DBP exposure delayed postnatal resumption of GC proliferation, leading to bigger deficits in numbers, and delayed re-expression of DMRT1 and radial GC migration. Therefore, DBP differentially affects foetal GC in rats according to stage of gestation, effects that may be relevant to the human because of their nature (OCT4, DMRT1 effects) or because similar effects are demonstrable in vitro on human foetal testes (GC number). Identification of the mechanisms underlying these effects could give a new insight into environment-sensitive mechanisms in early foetal GC development that could potentially be relevant to TGCC origins.

Sertoli cell numbers and spermatogenic efficiency are increased in inducible nitric oxide synthase mutant mice.

Nitric oxide (NO) is produced via oxidation of l-arginine by nitric oxide synthases (NOSs), and is known as inducible (iNOS), neuronal, endothelial or testis-specific. Suggesting important functions for NOS in the normal rat and mouse testis, iNOS is reported to be constitutively expressed in Leydig cells (LC), Sertoli cells (SC) and germ cells. In our study, we sought to provide further insights into the roles of iNOS in the adult mouse testis using iNOS(-/-) mice. Perfusion-fixed testes from wild type (WT) and iNOS(-/-) mice were used for histological and stereological evaluations. Some of the mice had been injected with (3) H-thymidine to label proliferating cells and to determine the duration of spermatogenesis that was unaffected in iNOS(-/-) mice. Both LC nuclear volume and individual cell size were significantly decreased in iNOS(-/-) mice, but the total number of LC per testis was increased (p < 0.05) by approximately 16%. The number of SC per testis was strikingly increased (approximately twofold) in iNOS(-/-) mice, and testis weight and DSP per gram of testis (spermatogenic efficiency) were similarly increased. The anogenital distance was also significantly increased in iNOS(-/-) mice, and this key endpoint suggests that the augmentation observed for the SC number may be related to increased foetal T-exposure during the masculinization programming window. Compared with WT testes, the numbers of spermatocytes and spermatids and SC per tubule cross sections were significantly increased in iNOS(-/-) mice. Except for stages V-VI and VII-VIII, iNOS(-/-) mice exhibited approximately 3.5-fold fewer apoptotic germ cells than in WT mice. Taken together, our results provide new evidence that iNOS plays an important role in numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survival and thus on daily sperm production.

Relative importance of prenatal and postnatal androgen action in determining growth of the penis and anogenital distance in the rat before, during and after puberty.

Experimental animal studies show that measurement of anogenital distance (AGD) and/or penis length may provide lifelong 'read-outs' of foetal androgen exposure during the masculinization programming window (MPW). However, variation in postnatal androgen exposure may complicate interpretation of such measurements. This is important to clarify if such measurements are to be applied to humans. The present aim was to evaluate effects of prenatal and/or postnatal manipulation of androgen production/action on growth of AGD and the penis in rats. Pregnant rats were treated daily before (e13.5-e21.5) and after birth (postnatal days 1-15) with either vehicle, 500 mg/kg di(n-butyl) phthalate (DBP) or 100 mg/kg flutamide (postnatal only) in prenatal + postnatal treatment combinations (N = 6 treatment combinations); DBP impairs androgen production whereas flutamide impairs androgen action. Male offspring were killed on postnatal day 8 (prepuberty), 25 (early puberty) or 90 (adulthood) when AGD was measured, the penis dissected out and its weight and length measured; plasma testosterone and ventral prostate weight were measured at day 90 to assess endogenous androgen exposure. In controls, penis length, girth and AGD increased 2.2-, 5.3-and 5.9-fold respectively from day 8 to day 90. Significant inhibition of penis growth and final length and girth was induced by treatments that inhibited postnatal androgen action. Conversely, growth and ultimate (adult) AGD was inhibited by prenatal inhibition of androgen production whereas postnatal androgen inhibition had negligible effect. Nevertheless, AGD and penis length were highly correlated at every age (R(2) > 0.33; p < 0.0001). However, altered endogenous androgen exposure may confound interpretation of changes in adults exposed prenatally/postnatally to DBP/flutamide. We conclude that AGD provides a lifelong guide to prenatal androgen exposure (in the MPW) whereas penis size reflects both prenatal + postnatal androgen exposure. At the group treatment level, prepubertal measurement of either AGD or penis size accurately predicts their size in adulthood.

Androgen action in the masculinization programming window and development of male reproductive organs.

We have shown previously that deficient androgen action within a masculinization programming window (MPW; e15.5-e18.5 in rats) is important in the origin of male reproductive disorders and in programming male reproductive organ size, but that androgen action postnatally may be important to achieve this size. To further investigate importance of the MPW, we used two rat models, in which foetal androgen production or action was impaired during the MPW by exposing in utero to either di(n-butyl) phthalate (DBP) or to flutamide. Reduced anogenital distance (AGD) was used as a monitor of androgen production/action during the MPW. Offspring were evaluated in early puberty (Pnd25) to establish if reproductive organ size was altered. The testes, penis, ventral prostate (VP) and seminal vesicles (SV) were weighed and penis length measured. Both DBP and flutamide exposure in the MPW significantly reduced penis, VP and SV size along with AGD at Pnd25; AGD and organ size were highly correlated. In DBP-, but not flutamide-, exposed animals, testis weight was also reduced and correlated with AGD. Intratesticular testosterone was also measured in control and DBP-exposed males during (e17.5) or after (e21.5) the MPW and related to AGD at e21.5. To evaluate the importance of postnatal androgen action in reproductive organ growth, the effect of combinations of prenatal and postnatal maternal treatments on AGD and penis size at Pnd25 was evaluated. In prenatally DBP-exposed animals, further postnatal exposure to either DBP or flutamide significantly reduced AGD and penis size in comparison with prenatal DBP exposure alone. In comparison, rats exposed postnatally to testosterone propionate after prenatal vehicle-exposure showed considerable increase in these parameters vs. controls. In conclusion, we show that the size of all male reproductive organs is programmed by androgen exposure in the MPW, but that growth towards this size is dependent on androgen action postnatally.