Epidemiology of Infantile Ureteropelvic Junction Obstruction in the US.

OBJECTIVE: To describe sex- and diagnosis-specific comorbidities, outcomes, and secular trends associated with ureteropelvic junction obstruction (UPJO) in a large, real-world population diagnosed with hydronephrosis in infancy. MATERIALS AND METHODS: We identified all infants ≤1 year old with ≥1 claim in the Optum Clinformatics 2007-2020 nationwide population database and used univariable and multivariable Cox regression analyses to estimate associations of demographic and clinical characteristics of infants with a UPJO diagnosis with surgical status. RESULTS: Of 22,349 infants with hydronephrosis (1.1% of infants; males-1.4%, females-0.7%), 1722 (7.7%; 7.9%-males, 7.2%-females) had UPJO. Follow-up was ≥1 year in 1198 (70%) and ≥3 years in 555 (32%) cases, and UPJO repair was performed in 542 children (31.5%; 32.3%-males, 29.5%-females); 77.7% within 1 year and 97.3% within 3 years. UPJO repair was associated with prior urinary tract infection (UTI) (hazard ratio (HR) 1.41, 95% confidence interval (CI) 1.12-1.76) and South (HR 1.42, 95% CI 1.14-1.78) or Midwest (HR 1.60, 95% CI 1.26-2.04) geographic region but did not change over time. CONCLUSION: This population-based study provides a real-world view of postnatally diagnosed hydronephrosis, focusing on UPJO, for which 522 cases (∼1/3) had ≥3 years continuous coverage. UPJO-associated comorbidities were more common in females, and the frequencies of UPJO-associated surgery and comorbidities were higher than in other studies. Other than UTI, no other associated kidney or urinary tract diagnoses were associated with UPJO repair. We identified unique sex- and diagnosis-specific differences in associated comorbidities and interventions in children diagnosed with UPJO in the first year of life.

Association of Obesity, Metabolic Syndrome, and Diabetes With Urinary Incontinence and Chronic Kidney Disease: Analysis of the National Health and Nutrition Examination Survey, 2003-2020.

PURPOSE: Diabetes and obesity, components of the metabolic syndrome (MetS), are risk factors for urinary incontinence (UI) and chronic kidney disease (CKD). We interrogated US population-based data to explore independent, sex-specific associations between nondiabetic MetS, with and without obesity, and UI and/or CKD. MATERIALS AND METHODS: We analyzed data from 8586 males and 8420 females ≥20 years from the National Health and Nutrition Examination Survey. Multivariable logistic regression models were used to examine associations of UI or CKD with diabetes and 4 nondiabetic obesity/metabolic phenotypes: non-MetS/nonobese, MetS/nonobese, non-MetS/obese, and MetS/obese. Multinominal logistic regression models were used to assess associations of co-occurring UI/CKD with obesity/metabolic phenotypes. RESULTS: Male MetS/obese participants had increased odds of any UI (1.25; 95% CI 1.00-1.57) and urgency UI (1.36; 1.03-1.80), compared with non-MetS/nonobese participants. Female MetS/obese participants had increased odds of any UI (2.16; 95% CI 1.76-2.66), stress UI (1.51; 1.21-1.87), and mixed UI (1.66; 1.31-2.11) compared with non-MetS/nonobese participants. The odds of co-occurring UI/CKD were increased relative to either condition alone in persons with diabetes, and in males with MetS/obese phenotypes and females with MetS phenotypes as compared to same sex participants with neither obesity nor MetS. CONCLUSIONS: We found novel associations between MetS/obese and urgency UI in males without diabetes, and between SUI and both MetS and obesity in females without diabetes. Odds estimates for UI/CKD were increased by existing obesity or MetS as compared to those for UI or CKD alone. Improved understanding of modifiable factors associated with UI will inform prevention and treatment opportunities.

The RISE FOR HEALTH study: Methods for in-person assessment and biospecimen collection.

OBJECTIVES: To describe the methods for the in-person assessment of the RISE FOR HEALTH (RISE) study, a population-based multicenter prospective cohort study designed to identify factors that promote bladder health and/or prevent lower urinary tract symptoms in adult women, conducted by the Prevention of Lower Urinary Tract Symptoms Research Consortium (PLUS). METHODS AND RESULTS: A subset of RISE participants who express interest in the in-person assessment will be screened to ensure eligibility (planned n = 525). Eligible consenting participants are asked to complete 15 physical assessments in addition to height and weight, to assess pelvic floor muscle function, musculoskeletal (MSK) status, and pain, and to provide urogenital microbiome samples. Pelvic floor muscle assessments include presence of prolapse, strength, levator attachment integrity (tear) and myofascial pain. MSK tests evaluate core stability, lumbar spine, pelvic girdle and hip pain and function. Participants are asked to complete the Short Physical Performance Battery to measure balance, lower extremity strength, and functional capacity. All participants are asked to provide a voided urine sample and a vaginal swab for microbiome analyses; a subset of 100 are asked to contribute additional samples for feasibility and validation of a home collection of urinary, vaginal, and fecal biospecimens. RESULTS: Online and in-person training sessions were used to certify research staff at each clinical center before the start of RISE in-person assessments. Standardized protocols and data collection methods are employed uniformly across sites. CONCLUSIONS: The RISE in-person assessment is an integral portion of the overall population-based RISE study and represents an innovative approach to assessing factors hypothesized to promote bladder health and/or prevent lower urinary tract symptoms. Data collected from this assessment will be used to prioritize future research questions and prevention strategies and interventions. This description of the assessment methods is intended to provide methodologic transparency and inform other researchers who join efforts to understand and improve bladder health.

RISE FOR HEALTH: Rationale and protocol for a prospective cohort study of bladder health in women.

INTRODUCTION: The spectrum of bladder health and the factors that promote bladder health and prevent lower urinary tract symptoms (LUTS) among women are not well understood. This manuscript describes the rationale, aims, study design, sampling strategy, and data collection for the RISE FOR HEALTH (RISE) study, a novel study of bladder health in women conducted by the Prevention of Lower Urinary Tract Symptom (PLUS) Research Consortium. METHODS AND RESULTS: RISE is a population-based, multicenter, prospective longitudinal cohort study of community-dwelling, English- and Spanish-speaking adult women based in the United States. Its goal is to inform the distribution of bladder health and the individual factors (biologic, behavioral, and psychosocial) and multilevel factors (interpersonal, institutional, community, and societal) that promote bladder health and/or prevent LUTS in women across the life course. Key study development activities included the: (1) development of a conceptual framework and philosophy to guide subsequent activities, (2) creation of a study design and sampling strategy, prioritizing diversity, equity, and inclusion, and (3) selection and development of data collection components. Community members and cross-cultural experts shaped and ensured the appropriateness of all study procedures and materials. RISE participants will be selected by simple random sampling of individuals identified by a marketing database who reside in the 50 counties surrounding nine PLUS clinical research centers. Participants will complete self-administered surveys at baseline (mailed paper or electronic) to capture bladder health and LUTS, knowledge about bladder health, and factors hypothesized to promote bladder health and prevent LUTS. A subset of participants will complete an in-person assessment to augment data with objective measures including urogenital microbiome specimens. Initial longitudinal follow-up is planned at 1 year. DISCUSSION: Findings from RISE will begin to build the necessary evidence base to support much-needed, new bladder health promotion and LUTS prevention interventions in women.

Regional Variation in Androgen Receptor Expression and Biomechanical Properties May Contribute to Cryptorchidism Susceptibility in the LE/orl Rat.

Background: The process of testicular descent requires androgen and insulin-like 3, hormones secreted by fetal Leydig cells. Knowledge concerning distinct and common functions of these hormones in regulating development of the fetal gubernaculum remains limited and/or conflicting. The current studies were designed to better define characteristics of androgen receptor (AR) expression, function and regulation, as well as the biomechanical properties of normal and cryptorchid gubernaculum during fetal development. Methods: We studied fetal gubernacula from Long Evans outbred (LE/wt) rats and an inbred (LE/orl) strain with an inherited form of cryptorchidism associated with an AR signaling defect. Gubernacular cells or whole organs obtained from LE/wt and LE/orl fetal gubernacula underwent AR immunostaining and quantitative image analysis. The effects of dihydrotestosterone (DHT) on AR expression, muscle fiber morphology, hyaluronan (HA) levels and glycosaminoglycan (GAG) content were measured in LE/wt gubernacula. Finally, the spatial mechanics of freshly harvested LE/wt and LE/orl fetal gubernacula were compared using micropipette aspiration. Results: AR is expressed in the nucleus of mesenchymal core, tip and cord cells of the embryonic (E) day 17 and 21 fetal gubernaculum, and is enhanced by DHT in primary cultures of gubernacular mesenchymal cells. Enhanced AR expression at the tip was observed in LE/wt but not LE/orl gubernacula. In in vitro studies of whole mount fetal gubernaculum, DHT did not alter muscle fiber morphology, HA content or GAG production. Progressive swelling with reduced cellular density of the LE/wt gubernaculum at E19-21 was associated with increased central stiffness in LE/wt but not in LE/orl fetuses. Conclusions: These data confirm nuclear AR expression in gubernacular mesenchyme with distal enhancement at the tip/cord region in LE/wt but not LE/orl rat fetuses. DHT enhanced cellular AR expression but had no major effects on muscle morphology or matrix composition in the rat fetal gubernaculum in vitro. Regional increased stiffness and decreased cell density between E19 and E21 were observed in LE/wt but not LE/orl fetal gubernacula. Developmental differences in cell-specific AR expression in LE/orl fetal gubernacula may contribute to the dysmorphism and aberrant function that underlies cryptorchidism susceptibility in this strain.

Perspective: A Neuro-Hormonal Systems Approach to Understanding the Complexity of Cryptorchidism Susceptibility.

Nonsyndromic cryptorchidism is a common multifactorial, condition with long-term risks of subfertility and testicular cancer. Revealing the causes of cryptorchidism will likely improve prediction and prevention of adverse outcomes. Herein we provide our current perspective of cryptorchidism complexity in a synthesis of cumulative clinical and translational data generated by ourselves and others. From our recent comparison of genome-wide association study (GWAS) data of cryptorchidism with or without testicular germ cell tumor, we identified RBFOX family genes as candidate susceptibility loci. Notably, RBFOX proteins regulate production of calcitonin gene-related peptide (CGRP), a sensory neuropeptide linked to testicular descent in animal models. We also re-analyzed existing fetal testis transcriptome data from a rat model of inherited cryptorchidism (the LE/orl strain) for enrichment of Leydig cell progenitor genes. The majority are coordinately downregulated, consistent with known reduced testicular testosterone levels in the LE/orl fetus, and similarly suppressed in the gubernaculum. Using qRT-PCR, we found dysregulation of dorsal root ganglia (DRG) sensory transcripts ipsilateral to undescended testes. These data suggest that LE/orl cryptorchidism is associated with altered signaling in possibly related cell types in the testis and gubernaculum as well as DRG. Complementary rat and human studies thus lead us to propose a multi-level, integrated neuro-hormonal model of testicular descent. Variants in genes encoding RBFOX family proteins and/or their transcriptional targets combined with environmental exposures may disrupt this complex pathway to enhance cryptorchidism susceptibility. We believe that a systems approach is necessary to provide further insight into the causes and consequences of cryptorchidism.

Cryptorchidism in Boys With Cerebral Palsy Is Associated With the Severity of Disease and With Co-Occurrence of Other Congenital Anomalies.

BACKGROUND: Cryptorchidism is reported in 40-50% of small case series of cerebral palsy (CP) and attributed to hypothalamic-pituitary-gonadal axis abnormalities, intellectual disability (ID), or cremaster spasticity. We collected demographic and clinical data to define the frequency of cryptorchidism and clinical comorbidities in a large CP population. METHODS: Electronic health record data were collected for all male patients ≥7 years of age seen in a large, multidisciplinary CP clinic between 2000 and 2016. Variables including age, testicular position, surgical findings, CP severity, birth history, and comorbidities were tested for association using univariable and stepwise backward logistic regression analyses. RESULTS: Of 839 established patients, testis position was scrotal in 553, undescended in 185 (24%), retractile in 38 (5%), and undocumented in 63 cases. Cryptorchidism were diagnosed at a mean age of 5.8 years, with 20% documented as acquired, and testes were most commonly in the superficial inguinal pouch (41%) and associated with an inguinal hernia (56%). Severity was bilateral in 114/166 (69%) undescended and 24/36 (66%) retractile cases, respectively. Mean birth weight and the frequency of prematurity (55, 58, and 54%) and multiple birth (14, 13, and 9%) were not significantly different among the three groups. We observed a strong ordinal trend in the frequency of comorbidities, including quadriplegia, syndromic features/known genetic disease, intrauterine growth restriction (IUGR), death, brain malformations, seizures, gastrostomy, absent continence, ID and hearing, speech or visual impairment, with the retractile group holding the intermediate position for the majority. The stepwise multivariable analysis showed independent positive associations of cryptorchidism with quadriplegia, syndromic features/known genetic disease, hearing loss, and absent continence, and inverse associations with gestational age and multiple birth. CONCLUSION: These data suggest that cryptorchidism is less common than previously reported in CP cases, but most strongly associated with quadriplegia. Delayed diagnosis may be related to an acquired condition or to the multiple additional functional deficits that occur in this population. Our data suggest that UDT and CP may both be components of malformation syndromes occurring in singleton births whose clinical features are more likely to include earlier delivery, IUGR, hearing loss, and/or global spasticity.

Subphenotype meta-analysis of testicular cancer genome-wide association study data suggests a role for RBFOX family genes in cryptorchidism susceptibility.

STUDY QUESTION: Can subphenotype analysis of genome-wide association study (GWAS) data from subjects with testicular germ cell tumor (TGCT) provide insight into cryptorchidism (undescended testis, UDT) susceptibility? SUMMARY ANSWER: Suggestive intragenic GWAS signals common to UDT, TGCT case-case and TGCT case-control analyses occur in genes encoding RBFOX RNA-binding proteins (RBPs) and their neurodevelopmental targets. WHAT IS KNOWN ALREADY: UDT is a strong risk factor for TGCT, but while genetic risk factors for TGCT are well-known, genetic susceptibility to UDT is poorly understood and appears to be more complex. STUDY DESIGN, SIZE, DURATION: We performed a secondary subphenotype analysis of existing GWAS data from the Testicular Cancer Consortium (TECAC) and compared these results with our previously published UDT GWAS data, and with data previously acquired from studies of the fetal rat gubernaculum. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies from the National Cancer Institute (NCI), United Kingdom (UK) and University of Pennsylvania (Penn) that enrolled white subjects were the source of the TGCT GWAS data. We completed UDT subphenotype case-case (TGCT/UDT vs TGCT/non-UDT) and case-control (TGCT/UDT vs control), collectively referred to as 'TECAC' analyses, followed by a meta-analysis comprising 129 TGCT/UDT cases, 1771 TGCT/non-UDT cases, and 3967 unaffected controls. We reanalyzed our UDT GWAS results comprising 844 cases and 2718 controls by mapping suggestive UDT and TECAC signals (defined as P < 0.001) to genes using Ingenuity Pathway Analysis (IPA®). We compared associated pathways and enriched gene categories common to all analyses after Benjamini-Hochberg multiple testing correction, and analyzed transcript levels and protein expression using qRT-PCR and rat fetal gubernaculum confocal imaging, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: We found suggestive signals within 19 genes common to all three analyses, including RBFOX1 and RBFOX3, neurodevelopmental paralogs that encode RBPs targeting (U)GCATG-containing transcripts. Ten of the 19 genes participate in neurodevelopment and/or contribute to risk of neurodevelopmental disorders. Experimentally predicted RBFOX gene targets were strongly overrepresented among suggestive intragenic signals for the UDT (117 of 628 (19%), P = 3.5 × 10-24), TECAC case-case (129 of 711 (18%), P = 2.5 × 10-27) and TECAC case-control (117 of 679 (17%), P = 2 × 10-21) analyses, and a majority of the genes common to all three analyses (12 of 19 (63%), P = 3 × 10-9) are predicted RBFOX targets. Rbfox1, Rbfox2 and their encoded proteins are expressed in the rat fetal gubernaculum. Predicted RBFOX targets are also enriched among transcripts differentially regulated in the fetal gubernaculum during normal development (P = 3 × 10-31), in response to in vitro hormonal stimulation (P = 5 × 10-45) and in the cryptorchid LE/orl rat (P = 2 × 10-42). LARGE SCALE DATA: GWAS data included in this study are available in the database of Genotypes and Phenotypes (dbGaP accession numbers phs000986.v1.p1 and phs001349.v1p1). LIMITATIONS, REASONS FOR CAUTION: These GWAS data did not reach genome-wide significance for any individual analysis. UDT appears to have a complex etiology that also includes environmental factors, and such complexity may require much larger sample sizes than are currently available. The current methodology may also introduce bias that favors false discovery of larger genes. WIDER IMPLICATIONS OF THE FINDINGS: Common suggestive intragenic GWAS signals suggest that RBFOX paralogs and other neurodevelopmental genes are potential UDT risk candidates, and potential TGCT susceptibility modifiers. Enrichment of predicted RBFOX targets among differentially expressed transcripts in the fetal gubernaculum additionally suggests a role for this RBP family in regulation of testicular descent. As RBFOX proteins regulate alternative splicing of Calca to generate calcitonin gene-related peptide, a protein linked to development and function of the gubernaculum, additional studies that address the role of these proteins in UDT are warranted. STUDY FUNDING/COMPETING INTEREST(S): The Eunice Kennedy Shriver National Institute for Child Health and Human Development (R01HD060769); National Center for Research Resources (P20RR20173), National Institute of General Medical Sciences (P20GM103464), Nemours Biomedical Research, the Testicular Cancer Consortium (U01CA164947), the Intramural Research Program of the NCI, a support services contract HHSN26120130003C with IMS, Inc., the Abramson Cancer Center at Penn, National Cancer Institute (CA114478), the Institute of Cancer Research, UK and the Wellcome Trust Case-Control Consortium (WTCCC) 2. None of the authors reports a conflict of interest.

Modeling Adaptive and Nonadaptive Responses of Populations to Environmental Change.

Understanding how the natural world will be impacted by environmental change over the coming decades is one of the most pressing challenges facing humanity. Addressing this challenge is difficult because environmental change can generate both population-level plastic and evolutionary responses, with plastic responses being either adaptive or nonadaptive. We develop an approach that links quantitative genetic theory with data-driven structured models to allow prediction of population responses to environmental change via plasticity and adaptive evolution. After introducing general new theory, we construct a number of example models to demonstrate that evolutionary responses to environmental change over the short-term will be considerably slower than plastic responses and that the rate of adaptive evolution to a new environment depends on whether plastic responses are adaptive or nonadaptive. Parameterization of the models we develop requires information on genetic and phenotypic variation and demography that will not always be available, meaning that simpler models will often be required to predict responses to environmental change. We consequently develop a method to examine whether the full machinery of the evolutionarily explicit models we develop will be needed to predict responses to environmental change or whether simpler nonevolutionary models that are now widely constructed may be sufficient.

The emergence of longevous populations.

The human lifespan has traversed a long evolutionary and historical path, from short-lived primate ancestors to contemporary Japan, Sweden, and other longevity frontrunners. Analyzing this trajectory is crucial for understanding biological and sociocultural processes that determine the span of life. Here we reveal a fundamental regularity. Two straight lines describe the joint rise of life expectancy and lifespan equality: one for primates and the second one over the full range of human experience from average lifespans as low as 2 y during mortality crises to more than 87 y for Japanese women today. Across the primate order and across human populations, the lives of females tend to be longer and less variable than the lives of males, suggesting deep evolutionary roots to the male disadvantage. Our findings cast fresh light on primate evolution and human history, opening directions for research on inequality, sociality, and aging.

Genomic copy number variation association study in Caucasian patients with nonsyndromic cryptorchidism.

BACKGROUND: Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population. METHODS: Genome wide genotyping were performed in 559 cases and 1772 controls (Group 1) using Illumina HumanHap550 v1, HumanHap550 v3 or Human610-Quad platforms and in 353 cases and 1149 controls (Group 2) using the Illumina Human OmniExpress 12v1 or Human OmniExpress 12v1-1. Signal intensity data including log R ratio (LRR) and B allele frequency (BAF) for each single nucleotide polymorphism (SNP) were used for CNV detection using PennCNV software. After sample quality control, gene- and CNV-based association tests were performed using cleaned data from Group 1 (493 cases and 1586 controls) and Group 2 (307 cases and 1102 controls) using ParseCNV software. Meta-analysis was performed using gene-based test results as input to identify significant genes, and CNVs in or around significant genes were identified in CNV-based association test results. Called CNVs passing quality control and signal intensity visualization examination were considered for validation using TaqMan CNV assays and QuantStudio® 3D Digital PCR System. RESULTS: The meta-analysis identified 373 genome wide significant (p < 5X10-4) genes/loci including 49 genes/loci with deletions and 324 with duplications. Among them, 17 genes with deletion and 1 gene with duplication were identified in CNV-based association results in both Group 1 and Group 2. Only 2 genes (NUCB2 and UPF2) containing deletions passed CNV quality control in both groups and signal intensity visualization examination, but laboratory validation failed to verify these deletions. CONCLUSIONS: Our data do not support that structural variation is a major cause of nonsyndromic cryptorchidism.

Genetic, Maternal, and Environmental Risk Factors for Cryptorchidism: An Update.

Unilateral or bilateral cryptorchidism is an isolated anomaly in the majority of cases, with evidence to date suggesting that it is a complex disorder resulting from interactions between genetic and environmental factors. Population, family, and limited genome-wide association data suggest moderate genetic risk, multiple susceptibility loci, and a role for the maternal environment. Epidemiologic studies have identified low birth weight or intrauterine growth retardation as factors most strongly associated with cryptorchidism, with additional evidence suggesting that maternal smoking and gestational diabetes increase risk. Animal studies have shown that the testis regulates its own descent by secretion of hormones that stimulate differentiation of the gubernaculum, and that endocrine-disrupting chemicals (EDCs) exhibit antiandrogenic and/or estrogenic activity that alters testicular function or gubernacular response to hormonal stimulation. However, we have yet to determine the degree to which EDCs contribute to cryptorchidism risk in humans, in part due to the varying methodology used in epidemiological and exposure studies. Large populations will be required to define the gene-environment interactions that predispose to cryptorchidism, in view of multiple small effect genetic susceptibility loci, ubiquitous exposure to mixtures of EDCs, and possible epigenetic effects. The present review provides an update of potential genetic and environmental risk factors for cryptorchidism, and future work required to better understand the etiology of this common and complex disease.

Dead or gone? Bayesian inference on mortality for the dispersing sex.

Estimates of age-specific mortality are regularly used in ecology, evolution, and conservation research. However, estimating mortality of the dispersing sex, in species where one sex undergoes natal dispersal, is difficult. This is because it is often unclear whether members of the dispersing sex that disappear from monitored areas have died or dispersed. Here, we develop an extension of a multievent model that imputes dispersal state (i.e., died or dispersed) for uncertain records of the dispersing sex as a latent state and estimates age-specific mortality and dispersal parameters in a Bayesian hierarchical framework. To check the performance of our model, we first conduct a simulation study. We then apply our model to a long-term data set of African lions. Using these data, we further study how well our model estimates mortality of the dispersing sex by incrementally reducing the level of uncertainty in the records of male lions. We achieve this by taking advantage of an expert's indication on the likely fate of each missing male (i.e., likely died or dispersed). We find that our model produces accurate mortality estimates for simulated data of varying sample sizes and proportions of uncertain male records. From the empirical study, we learned that our model provides similar mortality estimates for different levels of uncertainty in records. However, a sensitivity of the mortality estimates to varying uncertainty is, as can be expected, detectable. We conclude that our model provides a solution to the challenge of estimating mortality of the dispersing sex in species with data deficiency due to natal dispersal. Given the utility of sex-specific mortality estimates in biological and conservation research, and the virtual ubiquity of sex-biased dispersal, our model will be useful to a wide variety of applications.

First trimester phthalate exposure and male newborn genital anomalies.

BACKGROUND: Anti-androgenic phthalates are environmental chemicals that affect male genital development in rodents leading to genitourinary birth defects. We examined whether first trimester phthalate exposure may exert similar effects in humans leading to an increased incidence of newborn male genital anomalies in a multi-center cohort study. METHODS: We recruited first trimester pregnant women within The Infant Development and the Environment Study (TIDES) from 2010 to 2012 from four study centers and limited analyses to all mother/male infant dyads who had complete urinary phthalate and birth exam data (N=371). We used multivariate logistic regression to determine the odds of having a genital anomaly in relation to phthalate exposure. RESULTS: Hydrocele was the primary abnormality observed in the cohort (N=30) followed by undescended testes (N=5) and hypospadias (N=3). We observed a statistically significant 2.5 fold increased risk (95% CI 1.1, 5.9) of having any anomaly and 3.0 fold increased risk (95% CI 1.2, 7.6) of isolated hydrocele in relation to a one log unit increase in the sum of di-ethylhexyl phthalate (DEHP) metabolites. CONCLUSIONS: First trimester urinary DEHP metabolite concentrations were associated with increased odds of any newborn genital anomaly, and this association was primarily driven by isolated hydrocele which made up the majority of anomalies in newborn males. The association with hydrocele has not been previously reported and suggests that it may be an endpoint affected by prenatal phthalate exposures in the first trimester of development. Future human studies should include hydrocele assessment in order to confirm findings.

Fetal Rat Gubernaculum Mesenchymal Cells Adopt Myogenic and Myofibroblast-Like Phenotypes.

PURPOSE: Gubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex. MATERIALS AND METHODS: Embryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle. RESULTS: PAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling with CellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle. CONCLUSIONS: Our data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.

Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat.

STUDY HYPOTHESIS: Susceptibility to inherited cryptorchidism in the LE/orl rat may be associated with genetic loci that influence developmental patterning of the gubernaculum by the fetal testis. STUDY FINDING: Cryptorchidism in the LE/orl rat is associated with a unique combination of homozygous minor alleles at multiple loci, and the encoded proteins are co-localized with androgen receptor (AR) and Leydig cells in fetal gubernaculum and testis, respectively. WHAT IS KNOWN ALREADY: Prior studies have shown aberrant perinatal gubernacular migration, muscle patterning defects and reduced fetal testicular testosterone in the LE/orl strain. In addition, altered expression of androgen-responsive, cytoskeletal and muscle-related transcripts in the LE/orl fetal gubernaculum suggest a role for defective AR signaling in cryptorchidism susceptibility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The long-term LE/orl colony and short-term colonies of outbred Crl:LE and Crl:SD, and inbred WKY/Ncrl rats were maintained for studies. Animals were intercrossed (LE/orl X WKY/Ncrl), and obligate heterozygotes were reciprocally backcrossed to LE/orl rats to generate 54 F2 males used for genotyping and/or linkage analysis. At least five fetuses per gestational time point from two or more litters were used for quantitative real-time RT-PCR (qRT-PCR) and freshly harvested embryonic (E) day 17 gubernaculum was used to generate conditionally immortalized cell lines. We completed genotyping and gene expression analyses using genome-wide microsatellite markers and single nucleotide polymorphism (SNP) arrays, PCR amplification, direct sequencing, restriction enzyme digest with fragment analysis, whole genome sequencing (WGS), and qRT-PCR. Linkage analysis was performed in Haploview with multiple testing correction, and qRT-PCR data were analyzed using ANOVA after log transformation. Imaging was performed using custom and commercial antibodies directed at candidate proteins in gubernaculum and testis tissues, and gubernaculum cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: LE/orl rats showed reduced fertility and fecundity, and higher risk of perinatal death as compared with Crl:LE rats, but there were no differences in breeding outcomes between normal and unilaterally cryptorchid males. Linkage analysis identified multiple peaks, and with selective breeding of outbred Crl:LE and Crl:SD strains for alleles within two of the most significant (P < 0.003) peaks on chromosomes 6 and 16, we were able to generate a non-LE/orl cryptorchid rat. Associated loci contain potentially functional minor alleles (0.25-0.36 in tested rat strains) including an exonic deletion in Syne2, a large intronic insertion in Ncoa4 (an AR coactivator) and potentially deleterious variants in Solh/Capn15, Ankrd28, and Hsd17b2. Existing WGS data indicate that homozygosity for these combined alleles does not occur in any other sequenced rat strain. We observed a modifying effect of the Syne2(del) allele on expression of other candidate genes, particularly Ncoa4, and for muscle and hormone-responsive transcripts. The selected candidate genes/proteins are highly expressed, androgen-responsive and/or co-localized with developing muscle and AR in fetal gubernaculum, and co-localized with Leydig cells in fetal testis. LIMITATIONS, REASONS FOR CAUTION: The present study identified multiple cryptorchidism-associated linkage peaks in the LE/orl rat, containing potentially causal alleles. These are strong candidate susceptibility loci, but further studies are needed to demonstrate functional relevance to the phenotype. WIDER IMPLICATIONS OF THE FINDINGS: Association data from both human and rat models of spontaneous, nonsyndromic cryptorchidism support a polygenic etiology of the disease. Both the present study and a human genome-wide association study suggest that common variants with weak effects contribute to susceptibility, and may exist in genes encoding proteins that participate in AR signaling in the developing gubernaculum. These findings have potential implications for the gene-environment interaction in the etiology of cryptorchidism. LARGE SCALE DATA: Sequences were deposited in the Rat Genome Database (RGD, http://rgd.mcw.edu/). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by: R01HD060769 from the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD), 2P20GM103446 and P20GM103464 from the National Institute of General Medical Sciences (NIGMS), and Nemours Biomedical Research. The authors have no competing interests to declare.

Pathway analysis supports association of nonsyndromic cryptorchidism with genetic loci linked to cytoskeleton-dependent functions.

STUDY QUESTION: What are the genetic loci that increase susceptibility to nonsyndromic cryptorchidism, or undescended testis? SUMMARY ANSWER: A genome-wide association study (GWAS) suggests that susceptibility to cryptorchidism is heterogeneous, with a subset of suggestive signals linked to cytoskeleton-dependent functions and syndromic forms of the disease. WHAT IS KNOWN ALREADY: Population studies suggest moderate genetic risk of cryptorchidism and possible maternal and environmental contributions to risk. Previous candidate gene analyses have failed to identify a major associated locus, although variants in insulin-like 3 (INSL3), relaxin/insulin-like family peptide receptor 2 (RXFP2) and other hormonal pathway genes may increase risk in a small percentage of patients. STUDY DESIGN, SIZE, DURATION: This is a case-control GWAS of 844 boys with nonsyndromic cryptorchidism and 2718 control subjects without syndromes or genital anomalies, all of European ancestry. PARTICIPANTS/MATERIALS, SETTING, METHODS: All boys with cryptorchidism were diagnosed and treated by a pediatric specialist. In the discovery phase, DNA was extracted from tissue or blood samples and genotyping performed using the Illumina HumanHap550 and Human610-Quad (Group 1) or OmniExpress (Group 2) platform. We imputed genotypes genome-wide, and combined single marker association results in meta-analyses for all cases and for secondary subphenotype analyses based on testis position, laterality and age, and defined genome-wide significance as P = 7 × 10(-9) to correct for multiple testing. Selected markers were genotyped in an independent replication group of European cases (n = 298) and controls (n = 324). We used several bioinformatics tools to analyze top (P < 10(-5)) and suggestive (P < 10(-3)) signals for significant enrichment of signaling pathways, cellular functions and custom gene lists after multiple testing correction. MAIN RESULTS AND THE ROLE OF CHANCE: In the full analysis, we identified 20 top loci, none reaching genome-wide significance, but one passing this threshold in a subphenotype analysis of proximal testis position (rs55867206, near SH3PXD2B, odds ratio = 2.2 (95% confidence interval 1.7, 2.9), P = 2 × 10(-9)). An additional 127 top loci emerged in at least one secondary analysis, particularly of more severe phenotypes. Cytoskeleton-dependent molecular and cellular functions were prevalent in pathway analysis of suggestive signals, and may implicate loci encoding cytoskeletal proteins that participate in androgen receptor signaling. Genes linked to human syndromic cryptorchidism, including hypogonadotropic hypogonadism, and to hormone-responsive and/or differentially expressed genes in normal and cryptorchid rat gubernaculum, were also significantly overrepresented. No tested marker showed significant replication in an independent population. The results suggest heterogeneous, multilocus and potentially multifactorial susceptibility to nonsyndromic cryptorchidism. LIMITATIONS, REASONS FOR CAUTION: The present study failed to identify genome-wide significant markers associated with cryptorchidism that could be replicated in an independent population, so further studies are required to define true positive signals among suggestive loci. WIDER IMPLICATIONS OF THE FINDINGS: As the only GWAS to date of nonsyndromic cryptorchidism, these data will provide a basis for future efforts to understand genetic susceptibility to this common reproductive anomaly and the potential for additive risk from environmental exposures. STUDY FUNDING/COMPETING INTERESTS: This work was supported by R01HD060769 (the Eunice Kennedy Shriver National Institute for Child Health and Human Development (NICHD)), P20RR20173 (the National Center for Research Resources (NCRR), currently P20GM103464 from the National Institute of General Medical Sciences (NIGMS)), an Institute Development Fund to the Center for Applied Genomics at The Children's Hospital of Philadelphia, and Nemours Biomedical Research. The authors have no competing interests to declare.

Phenotype specific association of the TGFBR3 locus with nonsyndromic cryptorchidism.

PURPOSE: Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal. MATERIALS AND METHODS: We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum. RESULTS: We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum. CONCLUSIONS: These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFß signaling.