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.

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.

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.

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.

Altered infant feeding patterns in boys with acquired nonsyndromic cryptorchidism.

BACKGROUND: Genetic and environmental factors likely influence susceptibility to nonsyndromic cryptorchidism, a common disease presenting at birth or in later childhood. We compared cases and controls to define differential risk factors for congenital versus acquired cryptorchidism. METHODS: We compared questionnaire and clinical data from cases of congenital cryptorchidism (n = 230), acquired cryptorchidism (n = 182) and hernia/hydrocele (n = 104) with a group of healthy male controls (n = 358). Potential predictor variables (p < 0.2 in univariable analysis) were included in stepwise multivariable logistic regression models. RESULTS: Temporary (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.4-0.8) or exclusive (OR, 0.6; 95% CI, 0.4-0.9) breastfeeding was reduced and soy formula feeding increased (OR, 1.8; 95% CI, 1.2-2.9) in acquired but not congenital or hernia/hydrocele groups. The highest risk estimates were observed for primary soy formula feeding with limited or no breastfeeding (OR 2.5; 95% CI, 1.4-4.3; adjusted OR, 2.7; 95% CI, 1.4-5.4) in the acquired group. Primary feeding risk estimates were equivalent or strengthened when multivariable models were limited to age greater than 2 years, full-term or not small for gestational age, or Caucasian subjects. Pregnancy complications and increased maternal exposure to cosmetic or household chemicals were not consistently associated with either form of cryptorchidism in these models. CONCLUSIONS: Our data support reduced breastfeeding and soy formula feeding as potential risk factors for acquired cryptorchidism. Although additional studies are needed, hormonally active components of breast milk and soy formula could influence the establishment of normal testis position in the first months of life, leading to apparent ascent of testes in childhood. Birth Defects Research (Part A), 2012.

Reduced expression of androgen receptor and myosin heavy chain mRNA in cremaster muscle of boys with nonsyndromic cryptorchidism.

PURPOSE: To better define the developmental mechanisms of nonsyndromic cryptorchidism, we measured the expression of hormone receptor and muscle type specific mRNAs in target tissues of boys with and those without nonsyndromic cryptorchidism. MATERIALS AND METHODS: Prospectively collected cremaster muscle and/or hernia sac tissues from boys with congenital (79) or acquired (66) nonsyndromic cryptorchidism and hernia/hydrocele (controls, 84) were analyzed for hormone receptor (RXFP2, AR, ESR1, ESR2) and myosin heavy chain specific (MYH1, MYH2, MYH7) mRNA expression using real-time reverse transcriptase polymerase chain reaction. Log transformed mRNA, phenotype and feeding history data were statistically analyzed using Pearson's correlation, ANOVA and 2-sample t tests. RESULTS: AR mRNA expression was higher in cremaster muscle than in sac tissue, and significantly lower in congenital and acquired nonsyndromic cryptorchidism cases vs controls (p <0.01). Type 1 (slow/cardiac) MYH7 mRNA expression was also significantly reduced in both nonsyndromic cryptorchidism groups (p ≤ 0.002), while a reduction in type 2 (fast) MYH2 expression was more modest and significant only for the congenital cryptorchidism group (p <0.05). Cremasteric MYH7 and AR levels were strongly correlated (r(2) = 0.751, p <0.001). MYH7 and ESR1 mRNA levels were higher and lower, respectively, in boys with nonsyndromic cryptorchidism who were fed soy formula. Expression of other genes was not measurable. CONCLUSIONS: Our data suggest that boys with congenital and acquired nonsyndromic cryptorchidism differentially express AR and slow twitch specific MYH7 mRNA in the cremaster muscle, and that MYH7 expression is correlated with AR levels and soy formula use. These differences in gene expression may reflect aberrant hormonal signaling and/or innervation during development with the potential for secondary functional effects and failed testicular descent.

Disorders of sex differentiation: a pediatric urologist's perspective of new terminology and recommendations.

PURPOSE: In 2005 medical and lay experts convened (the Chicago Consensus), and reviewed and updated nomenclature and treatment recommendations in individuals with congenitally atypical gonadal, chromosomal or anatomical gender. This review summarizes, analyzes and considers the implications of these recommendations in pediatric urology practice. MATERIALS AND METHODS: Publications identified in a PubMed® search of 2000 to 2010 as well as relevant prior reports of new concepts and trends in the diagnosis of and treatment for intersex/ambiguous genitalia/disorders of sex differentiation, and responses to the Chicago Consensus were reviewed. RESULTS: In response to concerns regarding outdated, confusing and/or controversial terms, such as "intersex," "hermaphroditism" and "sex reversal," the consensus statement recommended a new taxonomy based on the umbrella term, "disorders of sex differentiation." Additional categorization based on sex chromosome complement was recommended but not clearly defined and variously interpreted. Routine use of multidisciplinary diagnostic and expert surgical teams, continuing psychosocial and psychosexual care, and full disclosure of alternatives relating to surgery type and timing were recommended. Early gender assignment was advocated but evidence-based guidance to support some aspects of care of affected individuals was insufficient. Pediatric urologists should remain abreast of new data refining the diagnoses and outcomes of disorders of sex differentiation, and ensure that their patients have access to multidisciplinary resources. CONCLUSIONS: Major changes in classification and expectations in the care of individuals with disorders of sex differentiation have occurred in recent years. Increasing focus on determining precise etiology and defining objective outcomes will help more clearly determine appropriate management and prognosis for this heterogeneous group of disorders.

The epidemiology of congenital cryptorchidism, testicular ascent and orchiopexy.

PURPOSE: The frequency, significance and possible etiology of testicular ascent (acquired cryptorchidism) are characterized in light of the known incidence and natural history of congenital cryptorchidism, and data provided by longitudinal and epidemiological studies of ascended testes and orchiopexy rates. MATERIALS AND METHODS: We comprehensively reviewed the literature addressing the epidemiology of congenital and acquired cryptorchidism and orchiopexy. RESULTS: The incidence of congenital cryptorchidism in full-term males at birth (2% to 4%) and at age 1 year (approximately 1%) has not increased in the last few decades. The risk of ascent may be as high as 50% in cases where 1 testis is significantly retractile. Ascended testes are typically unilateral (77%), identified in mid childhood and located distal to the inguinal canal (77%). Ascended and significantly retractile testes may be prone to the same germ cell maldevelopment seen in congenital cryptorchidism. Cumulative orchiopexy rates in defined populations are 2% to 4%, and mean age at orchiopexy remains higher than expected (greater than 4 years), despite a long held standard of care that includes recommendation for surgery by age 2. These data suggest that cryptorchidism may be acquired in a significant subset of cases. CONCLUSIONS: With close monitoring of young boys spontaneous ascent of testes from a scrotal to a suprascrotal position may be observed with time, due to either true or apparent testicular ascent, with possible adverse effects on germ cell development and fertility potential. Patients with significant testicular retractility appear to be at highest risk for acquired cryptorchidism, and should be followed closely at yearly intervals until puberty.