Insights into the Evolution of Spermatogenesis-Related Ubiquitin-Proteasome System Genes in Abdominal Testicular Laurasiatherians.

During embryonic development in mammals, the testicles generally descend into the scrotum, making the testicular temperature 2-4 °C lower than the core temperature via heat exchange and clearance, and thus more beneficial for normal spermatogenesis. Failure to descend, known as cryptorchidism, carries a series of risks such as infertility and testicular cancer. However, some mammals have evolved abdominal testes while maintaining healthy reproduction. To explore the underlying molecular mechanism, we conducted comparative genomic analyses and functional assays on the spermatogenesis-related ubiquitin-proteasome system (UPS) genes essential to sperm formation in representative laurasiatherians. Here, positive selection and rapid evolution of spermatogenesis-related UPS genes were identified in the abdominal testicular laurasiatherians. Moreover, potential convergent amino acids were found between distantly related species with similar abdominal testicles and functional analyses showed RNF8 (V437I) in abdominal testicular species (437I) has a stronger ubiquitination ability, which suggests that the mammals with abdominal testes might exhibit enhanced sperm cell histone clearance to maintain sperm formation. This evidence implies that, in response to "cryptorchidism injury", spermatogenesis-related UPS genes in the abdominal testicular species might have undergone adaptive evolution to stabilize sperm formation. Thus, our study could provide some novel insights into the reproductive adaptation in abdominal testicular mammals.

Structural and functional significance of scrotal ligament: a comparative histological study.

Gubernaculum testes is the most important parameter in testicular migration. At the end of migration, it is described as scrotal ligament, which has implications in testicular torsion. The present study aims to examine the structure of scrotal ligament and compare it with gubernaculum. Sixteen adult cadaveric testicular specimens and fourteen fetal testicular specimens of different age groups were examined after getting ethical clearance from the institute ethics committee and consent from the parents. Meticulous dissection was done. The length, site of proximal, and distal attachment of scrotal ligament and gubernaculum were noted and histologically evaluated. A separate scrotal ligament could not be delineated in any adult specimens. It merged with testicular coverings. Histological examination showed the presence of patchy areas of dense collagen fibres of variable density amidst loose areolar connective tissue. In contrast, fetal specimens showed the presence of a definitive gubernaculum testes and revealed the presence of mesenchymal tissue, collagen, elastic fibres, and myocytes which varied according to gestational age of fetuses. Structure of scrotal ligament and gubernaculum testes is highly variable. Description of scrotal ligament as a firm attachment from lower pole of testes to scrotum is controversial, questioning its role as protective factor in testicular torsion.

Fetal anogenital distance using ultrasound.

OBJECTIVE: This study measured anogenital distance (AGD) during late second/early third trimester of pregnancy to confirm previous findings that AGD can be measured noninvasively in the fetus using ultrasound and further showed differences in reference ranges between populations. METHOD: Two hundred ten singleton pregnancies were recruited at the Rosie Hospital, Cambridge, UK. A 2D ultrasound was performed between 26 and 30 weeks of pregnancy. AGD was measured from the centre of the anus to the base of the scrotum in males and to the posterior convergence of the fourchette in females. RESULTS: A significant difference in AGD between males and females (P < .0001) was found, replicating previous results with a significant correlation between estimated fetal weight (EFW) and AGD in males only (P = .006). A comparison of AGD using reference data from an Israeli sample (n = 118) and our UK sample (n = 208) showed a significant difference (P < .0001) in both males and females, after controlling for gestational age (GA). CONCLUSION: Our results confirm that AGD measurement in utero using ultrasound is feasible. In addition, there are strong sex differences, consistent with previous suggestions that AGD is influenced by prenatal androgen exposure. AGD lengths differ between the UK and Israel; therefore, population-specific normative values may be required for accurate clinical assessments.

Structural study of gubernaculum testis in fetuses with prune belly syndrome.

PURPOSE: We compared and contrasted the structure of the gubernaculum testis in fetuses with prune belly syndrome and normal controls. MATERIALS AND METHODS: We studied a total of 6 gubernacula from 3 male fetuses with prune belly syndrome and a total of 14 from 7 male fetuses without an anomaly. Gubernacular specimens were cut into 5 µm sections and stained with Masson trichrome to quantify connective tissue and smooth muscle cells, with Weigert stain to observe elastic fibers and with picrosirius red with polarization to observe collagen. Immunohistochemical analysis was done with tubulin to observe the nerves. Images were captured with a BX51 microscope and DP70 camera (Olympus®). Stereological analysis was done with Image-Pro and ImageJ (MediaCybernetics®) using a grid to determine volumetric density. Means were statistically compared with the Mann-Whitney test. All tests were 2-sided with p <0.05 considered statistically significant. RESULTS: Prune belly syndrome fetuses were at 17 to 31 weeks of gestation and control fetuses were at 12 to 35 weeks of gestation. Quantitative analysis showed no difference in the volumetric density of smooth muscle cells in prune belly syndrome vs control gubernacula (mean 15.70% vs 19%, p = 0.2321). Collagen fiber analysis revealed a predominance of green areas in prune belly syndrome gubernacula, suggesting collagen type III, and a predominance of red areas in control gubernacula, suggesting collagen type I. Elastic fibers were significantly smaller in prune belly syndrome gubernacula than in control gubernacula (mean 14.06% vs 24.6%, p = 0.0190). Quantitative analysis demonstrated no difference in the volumetric density of nerves in prune belly syndrome or control gubernacula (mean 5.200% vs 3.158%, p = 0.2302). CONCLUSIONS: The gubernaculum in fetuses with prune belly syndrome had altered concentrations of collagen and elastic fibers. These structural alterations could be one of the factors involved in cryptorchidism in prune belly syndrome.

The importance of the gubernaculum in testicular migration during the human fetal period

Objectives The objective of this review is to study the role of the gubernaculum in the testicular migration process during the human fetal period. Materials and Methods We performed a descriptive review of the literature about the role of the gubernaculum in testicular migration during the human fetal period. Results In the first phase of testicular migration, the gubernaculum enlarges to hold the testis near the groin and in the second phase the gubernaculum migrates across the pubic region to reach the scrotum. The proximal portion of the gubernaculum is attached to the testis and epididymis and the presence of multiple insertions in the distal gubernaculum is extremely rare. The presence of muscle and nerves in the human gubernaculum is very poor. The gubernaculum of patients with cryptorchidism has more fibrous tissue and less collagen and when the patients are submitted to hormonal treatment, the gubernaculum components alter significantly. Conclusions The gubernaculum presents significant structural modifications during testicular migration in human fetuses. .

Bifid scrotum and anocutaneous fistula associated with a perineal lipomatous tumor complicated by temporary bilateral cryptorchidism in utero mimicking ambiguous genitalia: 2-D/3-D fetal ultrasonography.

Ambiguous genitalia (AG) is a morphological diagnosis defined as genitalia not typical of a male or female. Findings mimicking AG, such as penoscrotal anomalies, anorectal malformations, and perineal lipomatous tumors, may prevent accurate identification of the fetal sex. We report a case of bifid scrotum and anocutaneous fistula associated with a perineal lipomatous tumor complicated by temporary bilateral cryptorchidism in utero, which were findings mimicking AG. Several perineal anomalies are associated developmental occurrences. In the present case, the combination of bifid scrotum and temporary bilateral cryptorchidism in the male fetus mimicked the combination of clitoromegaly and prominent labia, which are commonly observed in female fetuses. However, serial systemic assessments using prenatal 2-D/3-D ultrasonography and magnetic resonance imaging were unable to detect the anocutaneous fistula and differentiate the perineal lipomatous tumor. This case report suggests that the prenatal detection of perineal abnormalities may warn obstetricians of potentially undetected congenital perineal anomalies.

Testicular migration chronology: do the right and the left testes migrate at the same time? Analysis of 164 human fetuses.

OBJECTIVE: To determine if the right and the left testes migrate at the same time during the human fetal period. SUBJECTS AND METHODS: We studied 164 human fetuses (328 testes) ranging in age from 12 to 35 weeks post-conception. The fetuses were carefully dissected with the aid of a stereoscopic lens at ×16/25. The abdomen and pelvis were opened to identify and expose the urogenital organs. Testicular position was classified as: (a) Abdominal, when the testis was proximal to the internal ring; (b) Inguinal, when it was found between the internal and external inguinal rings); and (c) Scrotal, when it was inside the scrotum. RESULTS: The testes were abdominal in 71% of the cases, inguinal in 9.41%, and scrotal in 19.81%. There was asymmetry in testicular migration in nine cases (5.5%). In three of these nine cases, one testis was situated in the abdomen and the other in the inguinal canal; in another three one testis was situated in the abdomen and the other in the scrotum, and in the remaining three, one testis was in the inguinal canal and the other in the scrotum. In five of the nine cases of asymmetry, the right testis completed the migration first, but this was not statistically significant. CONCLUSION: Asymmetry in testicular migration is a rare event, accounting for <6% of the cases. The right testis seems to complete migration first.

The importance of the gubernaculum in testicular migration during the human fetal period.

OBJECTIVES: The objective of this review is to study the role of the gubernaculum in the testicular migration process during the human fetal period. MATERIALS AND METHODS: We performed a descriptive review of the literature about the role of the gubernaculum in testicular migration during the human fetal period. RESULTS: In the first phase of testicular migration, the gubernaculum enlarges to hold the testis near the groin and in the second phase the gubernaculum migrates across the pubic region to reach the scrotum. The proximal portion of the gubernaculum is attached to the testis and epididymis and the presence of multiple insertions in the distal gubernaculum is extremely rare. The presence of muscle and nerves in the human gubernaculum is very poor. The gubernaculum of patients with cryptorchidism has more fibrous tissue and less collagen and when the patients are submitted to hormonal treatment, the gubernaculum components alter significantly. CONCLUSIONS: The gubernaculum presents significant structural modifications during testicular migration in human fetuses.

Gubernaculum as icebreaker: do matrix metalloproteinases in rodent gubernaculum and inguinal fat pad permit testicular descent?

PURPOSE: Cryptorchidism is the most common male congenital abnormality. The rodent gubernaculum steers the testis from abdomen to scrotum postnatally by eversion and migration through the developing inguinal fat pad (IFP). We hypothesize that extracellular matrix remodeling in/around the gubernaculum is necessary for eversion and migration and is permitted by timed IFP maturation and aimed to examine regional development and matrix metalloproteinase (MMP) content. METHODS: Embryonic day 19 (E19) and postnatal days 0 and 2 (P0, P2) wild-type Sprague-Dawley rats (n = 10) were prepared for histologic examination (trichrome) and immunohistochemistry (membrane-type MMP-1 [MT1-MMP], MMP2) and analyzed using light/confocal microscopy. RESULTS: At E19, IFP contained fibroblasts and immature cells in an extensive collagenous extracellular matrix. Cells in the gubernaculum base were cytoplasmic-MT1-MMP-positive (inactive). At P0, the gubernaculum had everted, and adjacent cells were membranous-MT1-MMP-positive (active). At P2, the gubernaculum was migrating through the IFP, and adjacent cells were membranous-MT1-MMP-positive. Adipocyte maturation began cranially in the IFP and proceeded in a craniocaudal gradient until more uniformly mature at P2. CONCLUSION: The MT1-MMP-positive cells may remodel the gubernaculum for eversion and provide the collagenolysis necessary for migration, like an icebreaking ship, through the IFP, which matures to permit migration through collagen-rich tissue. Disruption of these processes may cause cryptorchidism.

The effect of flutamide on expression of androgen and estrogen receptors in the gubernaculum and surrounding structures during testicular descent.

BACKGROUND/PURPOSE: Inguinoscrotal testicular descent is controlled by androgens between embryonic days E16-19, but androgen receptor (AR) and estrogen receptor (ER) locations are unknown. We aimed to find AR, ERα, and ERß in the gubernaculum and inguinal fat pad (IFP) in normal rats and after flutamide treatment. METHODS: Sprague-Dawley timed-mated rats were injected with flutamide (75 mg/kg body weight/5% ethanol + oil) on E16-19 or vehicle alone. Male fetuses or pups (5-10/group) were collected at E16; E19; and postnatal (P) days 0, 2, 4, 8. Sections were prepared for hematoxylin and eosin or immunohistochemistry for AR, ERα, and ERß. Receptor labeling was quantitated as distinct nuclear labeling/100 µm(2) in gubernaculum and IFP. RESULTS: There was minimal gubernacular AR-labeling until E19, dramatically increasing postnatally. By contrast, at E16-E19 there was significant IFP AR immunoreactivity suppressed by flutamide (P < .05). No ERα expression was observed, but ERß was expressed in both gubernaculum and IFP, maximally at E16, but unchanged by flutamide. CONCLUSIONS: During the androgen sensitivity window (E16-19), the gubernaculum contains ERß but minimal ERα or AR, while the IFP, which is supplied by the genitofemoral nerve, contains abundant AR that are flutamide-sensitive. These results suggest that the IFP could be the site of androgenic action controlling gubernacular development.

Gone with the Wnt: the canonical Wnt signaling axis is present and androgen dependent in the rodent gubernaculum.

BACKGROUND/AIMS: How androgens control inguinoscrotal descent remains controversial but may include canonical Wnt signaling via the transcriptional co-activator ß-catenin. The canonical Wnt pathway transcribes genes regulating mesenchymal cell migration, fate, extracellular matrix remodeling, and in addition Axin2, a feedback product that reliably identifies Wnt activation. The relationship between ß-catenin and androgen receptor warranted investigation into the involvement of the canonical Wnt pathway in testicular descent. METHODS: Gubernacula from male Sprague-Dawley control (n = 22) and flutamide-treated (n = 18) rats at E17, E19, and D0 time-points were processed for immunohistochemistry. Sagittal sections stained for presence of androgen receptor, Axin2, and ß-catenin were analyzed by fluorescent confocal microscopy. RESULTS: At E19, ß-catenin was strongly expressed in the membrane of developing cremaster muscle cells and the cytoplasm of gubernacular core cells. Axin2 expression was ubiquitous in nuclei of gubernacular mesenchymal cells, representing canonical Wnt signaling. After androgen blockade, Axin2 was conspicuously absent in the fibroblasts of the gubernacular core while remaining unaffected elsewhere. Reduced staining of Axin2 in E17 and D0 gubernacula suggests that Wnt signaling coincides with androgen programming. CONCLUSION: Axin2 expression in the E19 gubernaculum confirms canonical Wnt pathway activation. Its absence in the core of flutamide-treated gubernacula indicates Wnt down-regulation. As androgen is required for inguinoscrotal descent, downstream Wnt signaling may control initial gubernacular remodeling. Defects in this complex molecular process may play a role in cryptorchidism.

Cremaster muscle myogenesis in the tip of the rat gubernaculum supports active gubernacular elongation during inguinoscrotal testicular descent.

PURPOSE: Cryptorchidism is a common abnormality and normal testicular descent is controlled by the gubernaculum. The cremaster may originate from abdominal muscles during gubernacular eversion or alternatively it may develop inside the gubernaculum. We studied cremaster myogenesis to determine how it develops. MATERIALS AND METHODS: Coronal sections of the pelvis were prepared from male Sprague-Dawley® rats and from males treated prenatally with the antiandrogen flutamide at embryonic day 19, and postnatal days 10, 19 and 35 after receiving ethical approval. Immunohistochemical stains were prepared for Ki67, Pax-7, myogenin, myosin heavy chain 7, Myh1, Myh2, Myh4, embryonic myosin, and slow and cardiac troponin T. Cell counts of the 1) gubernacular tip, 2) proximal gubernaculum/cremaster muscle and 3) adjacent abdominal wall are shown as a percent of positive fibers or positive cells per area. RESULTS: Throughout embryonic day 19, and postnatal days 10 and 19 proliferation (Ki67) was maximal at the gubernacular tip (p <0.001), as were muscle stem cells markers (Pax-7 p <0.05), early myogenesis (myogenin p <0.001) and immature muscle (Myh7, and slow and cardiac troponin T p <0.0001). In contrast, secondary (fast twitch, Myh1, 2 and 4) fibers were more common in abdominal muscles (p <0.0001). Differences in muscle maturity and composition decreased with time. Flutamide treated rats showed more cellular proliferation than controls postnatally on postnatal day 10 (p <0.001) as well as persistent immature embryonic myosin at the tip from postnatal day 19 (p <0.05). CONCLUSIONS: Results show that the rat cremaster muscle is more immature at the gubernacular tip, consistent with myogenesis occurring in the gubernaculum during migration to the scrotum, as proposed in humans.

Clear cells of Toker in the developing anogenital region of male and female fetuses.

The clear cells of Toker are a mysterious population of intra-epidermal glandular cells. They were originally described in nipples, but were recently observed in the vulva as well. It was hypothesized that intra-epidermal embryonic remnants or underlying glands were a potential source. The embryological aspects were investigated by studying specimens of the anogenital region of 18 male and 15 female fetuses between 12 and 39 weeks gestation. The search for Toker cells was enhanced by cytokeratin (CK) 7 immunohistochemistry. The investigation showed that Toker cell elements are a normal, though highly variable constituent of the developing anogenital region. The study revealed the following: (1) single intra-epidermal glandular vesicles near follicular anlages in interlabial sulcuses of female fetuses of 15 and 16.5 weeks gestation; (2) CK7+ solitary cells, clusters, and vesicles which were related to developing intra-epidermal follicular canal tracks and tended to disperse inside the epidermis in fetuses of approximately 18 weeks gestation; (3) dispersed CK7+ cells in fetuses of 19-23 weeks gestation; (4) characteristic CK7+ Toker cell proliferations in fetuses more than 23 weeks gestation. These observations indicate that in the anogenital region, primordial follicular cells programmed to participate in the formation of apocrine and mammary-like glands, become displaced into the epidermis where they disperse, and proliferate into Toker cell populations. However, the proximity of Toker cells to CK7+ cells in excretory ducts of late fetal apocrine and mammary-like glands suggested a possible additional source. Consequences for Toker cells of the breast and primary Paget disease are discussed.

Development of the gubernaculum during testicular descent in the rat.

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5-10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud.

How do you get six meters of epididymis inside a human scrotum?

It is very clear that the epididymis plays a crucial role in the maturation of spermatozoa, and without a fully developed and functional epididymis, male infertility will result. We are especially interested in understanding the mechanisms that regulate the development of this important organ because disruptions to epididymal function will also arise as a consequence of abnormal development. Very little is known either of the process of epididymal development or the nature and causes of congenital defects that lead to male infertility. A major event during Wolffian/epididymal duct embryonic development is elongation and coiling and this short review outlines potential mechanisms by which these events occur. It is hypothesized that elongation is the result of cell proliferation coupled with directed cell rearrangements, the later regulated by the planar cell polarity signaling pathway. Coiling proceeds in a proximal to distal manner, with three-dimensional coiling beginning approximately embryonic day 16.5 to 18.5 in the mouse. The exact mechanisms of coiling are not known but we hypothesize that it involves an interaction between the Wolffian duct epithelium and the surrounding mesenchyme cells, such that the extracellular matrix is remodeled to allow coiling and growth of the duct. Cell proliferation in the Wolffian duct appears to be dependent on the presence of androgens and mesenchymal factors during embryonic development, but lumicrine factors play an additional role during postnatal development.

Male sexual development in utero: testicular descent on prenatal magnetic resonance imaging.

OBJECTIVE: To visualize in utero male fetal testicular descent on magnetic resonance imaging (MRI) and to correlate it with gestational age. METHODS: This retrospective study included 202 MRI examination results of 199 male fetuses (17-39 gestational weeks) with normal anatomy or minor congenital abnormalities, following suspicion of anomalies on prenatal ultrasound examination. Using a 1.5-Tesla unit, multiplanar T2-weighted sequences were applied using a standard protocol to image and identify the scrotal content. The relative frequencies of unilateral and bilateral testicular descent were calculated and correlated with gestational age. RESULTS: Between 17 and 25 gestational weeks, neither unilateral nor bilateral testicular descent was visualized on MRI. Testicular descent was first observed at 25 + 4 weeks, in 7.7% of cases. 12.5% of 27-week fetuses showed unilateral descent and 50% showed bilateral descent. Bilateral descent was observed in 95.7% of cases, on average, from 30 to 39 weeks. CONCLUSIONS: Our results chart the time course of testicular descent on prenatal MRI, which may be helpful in the identification of normal male sexual development and in the diagnosis of congenital abnormalities, including the early detection of cryptorchidism.

The common fetal development of the mammary fat pad and gubernaculum.

BACKGROUND/AIMS: Recent work both from our laboratory and in marsupial models of testicular descent suggests a strong connection between the mammary line and gubernacular migration. This study investigated the relationship between the mammary fat pad (MFP) that underlies the mammary line and the developing gubernaculum by fluorescent immunohistochemistry. METHODS: Rats at E17 and E19 were fixed and processed for immunohistochemistry. Sagittal sections of male fetuses were stained with antibodies against androgen receptor (AR), prolyl-4 hydroxylase ß, Desmin, activated Notch-1, Jagged-1, and Ki-67. These were analyzed by fluorescent confocal microscopy. RESULTS: At E17 and E19, the MFP anlage forms a continuous distribution of fibroblasts passing immediately adjacent the gubernaculum to the future scrotum. Within this exists a distinct subpopulation of fibroblasts expressing AR distributed over the path of inguinoscrotal descent of the gubernaculum. Proliferation and Notch-1 signaling were similar throughout the MFP with differential Notch-1 signaling in the E19 gubernaculum. CONCLUSION: This investigation has identified the presence of a distinct AR-expressing subpopulation of MFP fibroblasts over the path of inguinoscrotal descent during the key androgenic programming window of this phase. This unique developmental pattern is consistent with a prime role for the MFP in testicular descent.

Hidden in plain sight: the mammary line in males may be the missing link regulating inguinoscrotal testicular descent.

BACKGROUND/PURPOSE: Inguinoscrotal testicular descent is controlled by androgens and the genitofemoral nerve, but the trigger for what makes the gubernaculum become a migratory organ like a limb bud remains unknown. Recent observations in the flutamide-treated rat suggested a link with the mammary line. We aimed, therefore, to reassess histologic anatomy in 2 different rodent models of androgen blockade, the testicular feminisation mouse (TFM) and the flutamide-treated rat. METHODS: Neonatal TFM mice and fetal and neonatal rats after pretreatment of dams with an antiandrogen, flutamide (75 mg/kg; sunflower oil; days 16-19), were prepared for histologic analysis of the inguinal region and compared with fetal and neonatal controls. RESULTS: Fetal control rats (E15.5 days) showed a mammary bud just outside the future inguinal canal adjacent to the gubernaculum. Neonatal TFM mice showed persistence of the inguinal breast bud supplied by the genitofemoral nerve. Flutamide-treated rats (D2) showed the gubernaculum surrounded by a persisting breast bud. CONCLUSIONS: The inguinal mammary line is adjacent to the gubernaculum in fetal rodents, and after androgen blockade, the gubernaculum becomes connected to the breast. The male mammary line, which is hidden in plain sight outside the inguinal canal, is made visible by androgen blockade. It may be the missing link in testicular descent, regulating gubernacular migration.

Completely separated scrotum and vesicointestinal fistula without exstrophy as a novel manifestation of aphallia: a case report.

A male infant born at 32 weeks of gestation and weighing 1.44 kg had penile agenesis, bifid scrotums, an anorectal anomaly, and suspected omphalocele. An operation revealed complete urethral agenesis, duplicated and blind bowels, a vesicointestinal fistula without exstrophy, and a urachal fistula; however, no omphaloceles were detected. To our knowledge, this is the first report on aphallia accompanied with completely separated scrotum and a vesicointestinal fistula without exstrophy; this condition is associated with urorectal septum malformation sequence (URSMS) and covered cloacal exstrophy. The possible embryogenesis is discussed in this study.