BDNF is upregulated by androgen in the inguinal fat pad of immature mice and may regulate inguinoscrotal testicular descent.

BACKGROUND: Androgens control rodent inguinoscrotal testicular descent during a "programming window" (E12-17). It is proposed that androgen masculinises the genitofemoral nerve, but the mechanism remains unknown. We investigate androgen receptor (AR)-containing target organs: inguinal fat pad (IFP) and mammary bud (MB), supplied by the genitofemoral nerve, hypothesizing that neurotrophic factors may retrogradely masculinise the GFN. METHODS: The IFP, MB and bulbocavernosus (BC) muscle were collected at E12.5/E17.5 from androgen receptor knockout (ARKO) mice and wild-type (WT) littermates. Immunofluorescence and gene expression (RT-qPCR; n = 8/group) for Bdnf, active (TrkB) and inactive (truncated TrkB) receptors, Cntf and Cntf receptor were performed. RESULTS: In the IFP at E12.5, ARKO TrkB mRNA expression was significantly downregulated compared to WT males (p < 0.0026). By E17.5, there was increased Bdnf expression (p < 0.0233). The MB had no differences at E12.5 and had regressed in WT males by E17.5. The BC had no differences at E12.5, but at E17.5 had significant upregulation of Bdnf expression in ARKO, compared to WT males. There were no differences in CNTF or CNTF receptor expression. CONCLUSIONS: Androgen alters active TrkB and Bdnf expression in the IFP. IFP Bdnf signalling may regulate "masculinisation" of the GFN sensory nerves to indirectly control inguinoscrotal testicular descent. IMPACT: Androgen mediates neurotrophin release in the inguinal fat pad in mice, which may facilitate normal testicular descent by masculinising the GFN by peripheral uptake of neurotrophin. This is the first study to examine the role of neurotrophins in testicular descent. This suggests novel steps in the mechanical process of normal testicular descent that may be abnormal in some children with undescended testes.

Testicular descent: A review of a complex, multistaged process to identify potential hidden causes of UDT.

BACKGROUND/PURPOSE: What causes normal descent of the testis in a fetus, and what goes wrong with this complex process to cause undescended testes (UDT), or cryptorchidism? Over the last 2 decades, most authors searching for the cause(s) of UDT have looked at the 2 main hormones involved, insulin-like hormone 3 (Insl3) and testosterone (T)/ dihydrotestosterone (DHT), and their known upstream (hypothalamic-pituitary axis) and intracellular 'downstream' pathways. Despite these detailed searches, the genetic causes of UDT remain elusive, which suggest the aetiology is multifactorial, and/or we are looking in the wrong place. METHODS: In this review we highlight the intricate morphological steps involved in testicular descent, which we propose may contain the currently 'idiopathic' causes of UDT. By integrating decades of research, we have underlined many areas that have been overlooked in the search for causes of UDT. RESULTS: It is quite likely that the common causes of UDT are still hidden in these areas, and we suggest examining these processes is worthwhile in the hope of finding the common genetic anomalies that lead to cryptorchidism. Given the fact that a fibrous barrier preventing descent is often described at orchidopexy, examination of the extracellular matrix enzymes needed to allow gubernacular migration may be a fruitful place to start. CONCLUSION: This review of the complex anatomical steps and hormonal regulation of testicular descent highlights many areas of morphology and signalling pathways that have been overlooked in the search for causes of UDT.

Frequency of inguinal herniotomy in Australia (1998-2017).

BACKGROUND/AIM: Closure of the processus vaginalis (PV) is considered as the last step of testicular descent. Therefore, patent processus vaginalis (PV), and inguinal hernias are linked to cryptorchidism. As the National Australian incidence of orchidopexy has decreased over the previous 20 years, we aimed to explore the incidence of inguinal herniotomy (including hydrocele) over time in Australia. METHODS: The National Department of Human Services (DHS) database, and Bureau of Statistics database were obtained for the years 1998-2017. The numbers of inguinal herniotomies in patients aged 0-4, 5-14 and 15-24 yearswere examined with ethical approval. RESULTS: Over the 20-year period, over 87,000 inguinal herniotomy procedures were performed in males. The incidence per year in males decreased across all ages over the 20-year period, but was most pronounced in infants and toddlers. Similar to males, the incidence in females decreased over time, with the ratio of procedures per head of population decreasing in children under 5 years of age. The ratio of male: females varied according to ages, and was between 2.8 and 6.2 males: 1 female. CONCLUSION: This study suggests that fewer 0-4-year olds are undergoing inguinal herniotomy, compared with 20 years ago. This is likely due to a change in practice for the management of unilateral symptomatic hernias, from routine bilateral herniotomies, to unilateral surgery. As well as less aggressive surgical intervention for hydroceles in boys. LEVEL OF EVIDENCE: III.

An immunohistochemical analysis of the effects of androgen receptor knock out on gubernacular differentiation in the mouse.

AIM: Cryptorchidism affects 2%-4% of newborn boys and causes infertility and cancer. While normal androgen function is required for successful inguinoscrotal descent, its exact role on gubernacular morphology remains unidentified. We aimed to decipher the effect of androgen blockade on the gubernaculum and surrounding structures. METHODS: Genetically modified mice with androgen receptor knock out (ARKO) were sectioned at ages E17, D0, and D2 for immunohistochemical analysis and D4 for morphological analysis (with ethical approval; A644). Mutants and control littermates were labeled with Ki67, Desmin, and Pax7 to identify the degree of gubernaculuar eversion and the composition of the growth center in the gubernaculum, using light or confocal microscopy. RESULTS: Androgen blockade prevented gubernacular eversion in all animals aged between E17 and D2 when compared to wild types. Furthermore, a growth center was visible, as indicated by a 'swirl' of immature fibroblasts, in D2 animals but was absent in ARKO mice. Moreover, the gubernacular cord was seen to increase in ARKO mice when compared to wild types and increased in size with age. There were no labeling differences in the antibodies tested for gubernacular differentiation. CONCLUSION: Gubernacular eversion in rodents prior to inguinoscrotal migration was androgen dependent, as well as maintenance of gubernacular cord length. This study shows that androgen blockade causes cryptorchidism in mice by preventing gubernacular eversion and possibly by preventing shortening of the gubernacular cord. Altering the morphology of the gubernaculum in response to androgen clearly contributes to the clinical problem of cryptorchidism.

Postnatal Germ Cell Development in the Cryptorchid Testis: The Key to Explain Why Early Surgery Decreases the Risk of Malignancy.

PURPOSE: Cryptorchidism is a risk factor for testicular malignancy and surgical treatment lowers this risk. This study aimed to investigate the germ cell behavior in prepubertal cryptorchid testes using immunohistochemical markers for germ cell malignancy to understand how early orchiopexy may possibly prevent cancer developing. MATERIALS AND METHODS: Histology sections from 1,521 consecutive testicular biopsies from 1,134 boys aged 1 month to 16.5 years operated for cryptorchidism were incubated with antibodies including antiplacental-like alkaline phosphatase (PLAP), anti-Oct3/4, anti-C-kit, and anti-D2-40. RESULTS: Oct3/4 and D2-40-positive germ cells are found throughout the first 2 years of life, with declining frequency thereafter. After 2 years, they should have disappeared and may indicate neoplasia. PLAP-positive cells were seen in 57 to 82% and C-kit-positive cells in 5 to 21% of cryptorchid testes between 4 and 13 years. Not until puberty did PLAP and C-kit-positive undifferentiated spermatogonial stem cells vanish. Only 0.3% of the present material had obvious prepubertal intratubular germ cell neoplasia (ITGCN) and they all had syndromic cryptorchidism. An additional three boys (0.3%) older than 2 years had weak Oct3/4 expression in undescended testes, but all cases were D2-40 negative. CONCLUSION: Prepubertal ITGCN was rare and mostly seen in syndromic cryptorchidism. In nonsyndromic cryptorchidism PLAP-positive undifferentiated spermatogonial stem cells persisted in a significant proportion of nontreated undescended testes and they will be especially sensitive to long-lasting abnormally high temperature that may be the single most important cause facilitating the accumulation of mutations during cell replication and the development of ITGCN to be prevented by orchiopexy.

Undescended testis: What paediatricians need to know.

Undescended testis (UDT) occurs when something goes wrong with testicular descent from high in the abdominal cavity to the scrotum. Normal descent occurs in two steps, with the transabdominal phase controlled by a new testicular hormone, insulin-like hormone 3, and the inguinoscrotal phase controlled by androgens. The latter phase requires a complex process of migration from the inguinal abdominal wall to the scrotum and is commonly defective, leading to the high incidence (2-4%) of UDT at birth. The clinical examination of babies and infants aims to confirm the persistence of congenital UDT by 3-6 months, so surgery can be optimally timed at 6-12 months. For those boys who develop acquired UDT later in childhood, the 'ascending' testis often needs surgery between 5 years and 10 years, so all boys should be screened again for UDT at school entry.

Frequency of revision orchidopexy in Australia 1995-2014.

BACKGROUND/AIM: International criteria currently suggest orchidopexy at 6-12months for congenital undescended testis (UDT). Some children require repeat orchidopexy for recurrent UDT. This study aimed to assess practice in Australia over a 20-year period. METHODS: We examined 20years of Australian orchidopexy data (1995-2014) from the Department of Human Services to explore the national revision orchidopexy rates over time. RESULTS: The total number of orchidopexy revisions was 890 over 20years compared with 25,984 primary operations. More than 50% of all primary and revision orchidopexies in 0-14year-old boys were performed in major population centers of NSW and Victoria (which hold 52% male population of same age), with a small number of revisions on 15-24year-old males. The incidence of revision orchidopexy significantly decreased over the 20-year period in boys ages 0-14years old, from 276 operations between 1995 and 1999 decreasing to 165 operations between 2010 and 2014 (-53%), compared to a population increase of +15% (p<0.05). CONCLUSION: These data demonstrate a decrease in revision orchidopexy since 1995, which may be related to change in referral practice with more children undergoing orchidopexy (primary and revision) by pediatric surgeons over the 20-year period. LEVEL OF EVIDENCE: Level IV. TYPE OF STUDY: Therapeutic Case Series with no Comparison Group.

Undescended testes: Diagnosis and timely treatment in Australia (1995-2014).

BACKGROUND: Routine primary care checks in infants and prepubertal boys aim for early detection and intervention of undescended testes (UDT). Congenital and acquired UDT cause infertility, and congenital UDT also increases testicular cancer risk. We examined 20 years of Australian orchidopexy data (1995-2014) to explore the national orchidopexy operation rates over time. METHODS: Orchidopexy and population data were collected from the Australian Bureau of Statistics (ABS) for 1995-2014, and census data for each age group were also collected. Poisson regressions were used to analyse the data. RESULTS: For patients aged DISCUSSION: The rate of orchidopexy per age has decreased in patients aged 5-14 years over the past 20 years, possibly indicating that acquired UDT is not being diagnosed and treated in some boys, risking infertility in adulthood.

What Animal Models of Testicular Descent and Germ Cell Maturation Tell Us about the Mechanism in Humans.

Testicular descent occurs in most mammals in two main steps that have different hormonal control and anatomical processes. The evolution of testicular descent reveals the same basic processes in humans and animals, with minor differences in timing and anatomy, especially the location of the scrotum and the processus vaginalis. Animal models are useful as they reveal some embryological processes that cannot be studies easily in humans, such as the potential role of the mammary line and the role of the genitofemoral nerve. Postnatal germ cell development is very similar in animal models and humans, except for the timing of arrival of the testis into the scrotum, which is before birth in humans versus around puberty in rodents. Once all the minor differences between animal models and humans are taken into account, animal experimentation has provided amazing insights into the mechanisms of testicular descent, and recently, how the postnatal germ cell develops in normally descended and undescended testes.

The undescended testis: Clinical management and scientific advances.

Undescended testes (UDT), where one or both testes fail to migrate to the base of the scrotum, can be congenital (2-5% of newborn males) or acquired (1-2% of males). The testis may be found in any position along its usual line of descent. Cryptorchidism affects the developing testicular germ cells and increases the risk of infertility and malignancy. Clinical management aims to preserve spermatogenesis and prevent the increased risk of seminoma. Examination to document the testicular position will guide the need for imaging, medical management and the surgical approach to orchidopexy.

Neurotrophin signaling in a genitofemoral nerve target organ during testicular descent in mice.

BACKGROUND/AIM: It has been proposed that androgens control inguinoscrotal testicular descent via release of calcitonin gene-related peptide (CGRP) from a masculinised genitofemoral nerve (GFN). As there are androgen receptors in the inguinoscrotal fat pad (IFP) during the window of androgen sensitivity (E14-17 in mouse embryos), we tested the hypothesis that neurotrophins in the IFP may masculinise the sensory fibers of the GFN supplying the gubernaculum and IFP prior to gubernacular migration. METHODS: Androgen-receptor knockout (ARKO) and wild-type (WT) mouse embryos were collected at E17, with ethical approval (AEC 734). Sagittal sections of IFP, mammary area and bulbocavernosus (BC) muscle were processed for standard histology and fluorescent immunohistochemistry for ciliary neurotrophic factor (CNTF), ciliary neurotrophic factor receptor (CNTFR) and cell nuclei (DAPI). RESULTS: In the ARKO mouse CNTFR immunoreactivity (CNTFR-IR) was increased in the IFP but decreased in BC. Perinuclear staining of CNTF-IR was seen in mouse sciatic nerve but only weakly in IFP. In the mammary area, also supplied by GFN, there were no differences in IR staining. CONCLUSION: This study found CNTFR-IR in the IFP was negatively regulated by androgen, suggesting that CNTF signaling may be suppressed in GFN sensory nerves to enable CGRP expression for regulating gubernacular migration in the male, but not the female. The indirect action of androgen via the GFN required for testicular descent may be one of the sites of anomalies in the putative multifactorial cause of cryptorchidism.

Molecular signals governing cremaster muscle development: clues for cryptorchidism.

BACKGROUND/AIM: Cryptorchidism affects 2-4% of newborn boys. Testicular descent requires the gubernaculum to differentiate into cremaster muscle (CM) during androgen-mediated inguino-scrotal descent, but the cellular mechanisms regulating this remodeling remain elusive. ß-Catenin, a marker of canonical Wnt signaling, promotes myogenic genes and cellular adhesion. We aimed to determine if androgen receptor (AR) blockade altered ß-catenin and its downstream myogenic proteins within the CM. METHOD: Gubernacula from male rats (n=12) and rats treated with anti-androgen, flutamide (n=12) at E19, D0, D2 were processed for immunohistochemistry. Antibodies against ß-catenin, embryonic myosin, and myogenin were visualized by confocal microscopy. RESULTS: At E19, ß-catenin immuno-reactivity (IR) localized to the CM membrane. By D2, cytoplasmic ß-catenin-IR was noted with overall ß-catenin-IR decreasing. Myogenic proteins resided primarily in cells containing ß-catenin on their plasma membrane. Embryonic myosin-IR was high at E19 and then decreased by D2, while myogenin-IR increased. AR blockade increased cytoplasmic ß-catenin at D2 and reduced levels of both myogenic proteins. CONCLUSION: Myogenic proteins are present in CM cells containing ß-catenin. AR blockade did not alter cellular adhesion via ß-catenin. In contrast, blocking AR prevented ß-catenin entering the nucleus and impaired CM myogenesis. Mutations in this pathway may result in idiopathic cryptorchidism.