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.

Modifications of erectile tissue components in the penis during the fetal period.

BACKGROUND: The penile erectile tissue has a complex microscopic anatomy with important functions in the mechanism of penile erection. The knowledge of such structures is necessary for understanding the normal physiology of the adult penis. Therefore, it is important to know the changes of these penile structures during fetal development. This study aims to analyze the development of the main components of the erectile tissue, such as collagen, smooth muscle fibers and elastic system fibers, in human fetuses. METHODOLOGY/PRINCIPAL FINDINGS: We studied the penises of 56 human fetuses aged 13 to 36 weeks post-conception (WPC). We used histochemical and immunohistochemical staining, as well as morphometric techniques to analyze the collagen, smooth muscle fibers and elastic system fibers in the corpus cavernosum and in the corpus spongiosum. These elements were identified and quantified as percentage by using the Image J software (NIH, Bethesda, USA). From 13 to 36 WPC, in the corpus cavernosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 19.88% to 36.60%, from 4.39% to 29.76% and from 1.91% to 8.92%, respectively. In the corpus spongiosum, the amount of collagen, smooth muscle fibers and elastic system fibers varied from 34.65% to 45.89%, from 0.60% to 11.90% and from 3.22% to 11.93%, respectively. CONCLUSIONS: We found strong correlation between the elements analyzed with fetal age, both in corpus cavernosum and corpus spongiosum. The growth rate of these elements was more intense during the second trimester (13 to 24 WPC) of gestation, both in corpus cavernosum and in corpus spongiosum. There is greater proportional amount of collagen in the corpus spongiosum than in corpus cavernosum during all fetal period. In the corpus spongiosum, there is about four times more collagen than smooth muscle fibers and elastic system fibers, during all fetal period studied.

Study of the ureter structure in anencephalic fetuses

Purpose The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. Materials and Methods We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). Results The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. Conclusions Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate ...

Study of the ureter structure in anencephalic fetuses.

PURPOSE: The objective of this paper is to analyze the structure of the ureter in normal and anencephalic human fetuses. MATERIALS AND METHODS: We studied 16 ureters from 8 human fetuses without congenital anomalies aged 16 to 27 weeks post-conception (WPC) and 14 ureters from 7 anencephalic fetuses aged 19 to 33 WPC. The ureters were dissected and embedded in paraffin, from which 5 µm thick sections were obtained and stained with Masson trichrome, to quantify smooth muscle cells (SMC) and to determine the ureteral lumen area, thickness and ureteral diameter. The samples were also stained with Weigert Resorcin Fucsin (to study elastic fibers) and Picro-Sirius Red with polarization and immunohistochemistry analysis of the collagen type III fibers to study collagen. Stereological analysis of collagen, elastic system fibers and SMC were performed on the sections. Data were expressed as volumetric density (Vv-%). The images were captured with an Olympus BX51 microscope and Olympus DP70 camera. The stereological analysis was done using the Image Pro and Image J programs. For biochemical analysis, samples were fixed in acetone, and collagen concentrations were expressed as micrograms of hydroxyproline per mg of dry tissue. Means were statistically compared using the unpaired t-test (p < 0.05). RESULTS: The ureteral epithelium was well preserved in the anencephalic and control groups. We did not observe differences in the transitional epithelium in the anencephalic and control groups. There was no difference in elastic fibers and total collagen distribution in normal and anencephalic fetuses. SMC concentration did not differ significantly (p = 0.1215) in the anencephalic and control group. The ureteral lumen area (p = 0.0047), diameter (p = 0.0024) and thickness (p = 0.0144) were significantly smaller in anencephalic fetuses. CONCLUSIONS: Fetuses with anencephaly showed smaller diameter, area and thickness. These differences could indicate that anencephalic fetal ureters tend to have significant structural alterations, probably due to cerebral lesions with consequent brain control damage of ureter nerves.

Estudo estrutural do pênis de fetos humanos normais e anencéfalos no segundo trimestre de gestação / Structural study of the penis of normal and anencephalic human fetuses in the second trimester of pregnancy

A anecencefalia é o Defeito do Tubo Neural (DTN) mais severo em fetos humanos. Há uma demanda crescente para reposição tissular em doenças crônicas e cirurgias reconstrutoras. Tecidos fetais têm sido utilizados como substitutos para órgãos sólidos. Comparar a estrutura e morfologia do corpo cavernoso e corpo esponjoso de pênis de fetos humanos anencéfalos e de controle a fim de propor um novo modelo para estudos biológicos e transplantes teciduais. Foram estudados 11 pênis de fetos de controle de 14 a 23 Semanas Pós Concepção (SPC), e cinco pênis de fetos anencéfalos de 18 a 22 SPC. Os órgãos foram removidos e processados pelas técnicas histo e imunohistoquímicas rotineiras. A análise do tecido conjuntivo, células musculares lisas e fibras elásticas foram realizadas em lâminas dos espécimes. Os dados foram expressos em Densidade de àrea (Da) utilizando-se um software de processamento digital. As médias foram comparadas utilizando-se o Teste - T não pareado e quando aplicável, a regressão linear simples foi utilizada. Foi considerada significância estatística se p<0,05. O septo intercavernoso encontrava-se presente em todas as amostras. Não foram observadas diferenças da Da do tecido colágeno e musculatura lisa dos pênis de fetos anencéfalos quando comparados aos normais. A regressão linear simples sugere que durante o desenvolvimento humano há um aumen2to gradual do tecido colágeno (R2=0,45) e uma diminuição da musculatura lisa (R =0,62) no corpo cavernoso de ambos os grupos. A elastina encontrava-se presente apenas em fetos a partir da 20ª SPC. Não houve diferença na estrutura da genitália entre fetos normais e enencéfalos. Apresença da elastina em fetos a partir da 20ª SPC é um dado objetivo da manutenção da capacidade de ereção nestes grupos. A histo e imunohistoquímica sugerem que o desenvolvimento do pênis destes fetos encontra-se inalterado. Futuros estudos deverão ser realizados com o objetivo de avaliar fetos anencéfalos como um potencial ...

Anencephaly is the most severe neural tube defect (NTD) in human fetuses. There is an increasing need for tissue replacement in chronic diseases and reconstructive surgeries. Fetal tissues have been used as a substitute for native organs. Compare the structure and morphology of the corpora cavernosa and spongiosum of penises from anencephalic and normal human fetuses to propose a new model for biological studies and tissue transplantation. We studied 11 penises from normal human fetuses, aged 14 to 23 weeks post-conception (WPC), and 5 penises from anencephalic fetuses, aged 18 to 22 WPC. The organs were removed and processed by routine histological and immunolabeling techniques. Analysis of connective tissue (Cot), smooth muscle (SMC) and elastic fibers (EF) were performed in sections. Data were expressed as area density (Ad) using digital processing and software. Means were statistically compared using the unpaired t test and linear regression was performed. Statistical significance was considered if p < 0.05. The Intracavernosal septum was present in all samples. We did not observe differences in the Ad of Cot and SMC in the penises of anencephalic fetuses when compared to normal ones. The simple linear regression suggested that during human development there is a gradual increase in Cot (R2= +0.45) and a decrease of SMC (R2=- 0.62) in the corpora cavernosa in both groups studied. Elastin was observed only in fetuses from 20th WPC. There was no difference in the structure of the corpora cavernosa and corpus spongiosum of anencephalic fetuses compared to normal ones. Elastin was documented from 20th WPC, which suggests the maintenance of erectile function. Histochemistry and immunolabeling suggested that penile shaft development is maintained and unaltered in anencephalic fetuses. Further studies should be performed to analyze anencephalic fetuses as a potential tissue donating group and a model for biological studies

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study.

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

The connective tissue of the adductor canal--a morphological study in fetal and adult specimens.

The adductor canal is a conical or pyramid-shaped pathway that contains the femoral vessels, saphenous nerve and a varying amount of fibrous tissue. It is involved in adductor canal syndrome, a claudication syndrome involving young individuals. Our objective was to study modifications induced by aging on the connective tissue and to correlate them to the proposed pathophysiological mechanism. The bilateral adductor canals and femoral vessels of four adult and five fetal specimens were removed en bloc and analyzed. Sections 12 microm thick were obtained and the connective tissue studied with Sirius Red, Verhoeff, Weigert and Azo stains. Scanning electron microscopy (SEM) photomicrographs of the surfaces of each adductor canal were also analyzed. Findings were homogeneous inside each group. The connective tissue of the canal was continuous with the outer layer of the vessels in both groups. The pattern of concentric, thick collagen type I bundles in fetal specimens was replaced by a diffuse network of compact collagen bundles with several transversal fibers and an impressive content of collagen III fibers. Elastic fibers in adults were not concentrated in the thick bundles but dispersed in line with the transversal fiber system. A dynamic compression mechanism with or without an evident constricting fibrous band has been proposed previously for adductor canal syndrome, possibly involving the connective tissue inside the canal. The vessels may not slide freely during movement. These age-related modifications in normal individuals may represent necessary conditions for this syndrome to develop.

The concentration of elastic fibres in the male urethra during human fetal development.

OBJECTIVE: To describe the distribution of elastic fibres in the developing male urethra and to provide stereological data of the concentration of elastic fibres in the human urethra. MATERIALS AND METHODS: Urethras were obtained from 10 fresh normal human fetuses at 15-36 weeks of gestation. A place-matched spongy urethra of a 27-year-old normal adult man was also analysed. Samples were fixed in Bouin's solution, embedded in paraffin and histologically processed. The elastic system fibres were evaluated by light microscopy using Weigert's resorcin-fuchsin technique after oxidation. Morphometric values were assessed by the point-counting method. The volumetric density (Vv) of elastic fibres was correlated with fetal age. RESULTS: At 15 weeks the elastic fibres were sparse and homogeneously distributed. The size and thickness of elastic fibres increased with age, mainly in the third trimester of gestation. Elastic fibres formed a randomly orientated network in the trabeculae of the corpus spongiosum. The mean (sem) Vv of elastic fibres in the spongy urethra was 5.2 (0.4)% in the fetus at 15 weeks and 14.8 (1.0)% at 36 weeks. In the urethra of the place- matched young man the Vv was 19.0 (1.3)%. The concentration of elastic fibres in the spongy urethra increased significantly with age. CONCLUSION: The high concentration of elastic fibres in the spongy urethra may partly explain its high extensibility. The progressive increase in elastic fibres during development implies functional adaptation of the fetal male urethra.

Cellular aspects of elastogenesis in the elastic tendon of the chicken wing.

Morphological, immunocytochemical and ultrastructural methods were used to investigate the role of cells during elastogenesis in the elastic tendon of the chicken wing. Intimate contact of the cell processes with elastic fibers was observed in adult birds. During development there was a sequential appearance of microfibril bundles that became progressively impregnated with amorphous elastin, which eventually predominated in fully developed elastic fibers. The growing elastic fibers were usually enveloped by recesses of the cell surface. The tendon cells were polarized in their association with fibrous components of the extracellular matrix. This arrangement suggests that these cells secrete and organize elastic and collagen fibers to different extracellular compartments. These results show that cells are intimately involved in producing components of different extracellular matrix fibers, in controlling their assembly, and in defining their borders and associations during development.

The development of fibrocartilage in the elastic tendon of the chicken wing.

The elastic tendon of the chicken wing has five morphologically distinct regions. One of these regions is a distally located fibrocartilage from which fibrous connections extend to the capsule of the distal radius. In adult birds, this region shows the characteristics of a tendon-compressed fibrocartilage, with an accumulation of proteoglycans between thick collagen bundles arranged in a basket-weave formation. Here we study the development of this fibrocartilage in order to of compare it with other tendon fibrocartilages and try to identify the factors involved in fibrocartilage differentiation. This fibrocartilage initially developed by cell enlargement and accumulation of vimentin, with simultaneous deposition of proteoglycans in the extracellular matrix and an increase in the amount and thickness of collagen bundles. Elastic fibers were minor components associated with the collagen bundles. Cells could be classified into two main types. One was typically fibrocartilaginous and the other was fibroblast-like, the latter occurring in close association with the collagen bundles. These results establish the steps in the development of the elastic tendon fibrocartilage and provide a basis for future studies.

Testicular migration: remodeling of connective tissue and muscle cells in human gubernaculum testis.

PURPOSE: We present the main morphological modifications in the human gubernaculum during testicular migration in humans. MATERIALS AND METHODS: We obtained 12 gubernacula from fresh, macroscopically normal human fetuses at 15 to 29 weeks of gestation. Collagen was evidenced using trichrome and Sirius red staining procedures, while Weigert's resorcinol-fuchsin and anti-human elastin antibody were used to reveal elastic system fibers. Smooth muscle cells were detected by anti-human smooth muscle alpha-actin antibody. RESULTS: When the testes were still located in the abdomen at 15 to 16 weeks of gestation, collagen fibers were sparse and embedded in a loose extracellular matrix. The amount of fibers then gradually increased with age and at 28 weeks of gestation the gubernaculum was mostly collagenous in composition. Elastic fibers had a similar growth pattern, although they were located mainly at the distal end of the gubernaculum. Fibroblasts largely predominated over other cell types and decreased in number with gestational age, whereas smooth muscle cells were restricted to the walls of blood vessels. Striated muscle cells were detected at the scrotal end of the gubernaculum, where they were disposed as isolated and scattered bundles running in various directions. Like fibroblasts, their number also decreased with age. CONCLUSIONS: During testicular migration gubernacular connective tissue undergoes extensive remodeling and ultimately becomes an essentially fibrous structure rich in collagen and elastic fibers. Such changes should decrease the size of the gubernaculum and, thus, contribute to other forces that cause the testes to move toward the scrotum. In fact, because of the lack of smooth muscle cells, and the amount and organization of striated muscle cells, active contraction of the gubernaculum is less likely to be an important factor in testicular descent.

Collagen and elastic fibers in the penis of human fetuses at 28 weeks postconception.

OBJECTIVES: The extracellular matrix is a key element in penile function and pathology, yet little is known of its development. Herein we investigated the morphological organization of collagen and elastin in the corpora cavernosa and tunica albuginea of human fetuses. MATERIAL AND METHODS: The penises from 5 fresh human fetuses at 28 weeks postconception (WPC) were routinely fixed and embedded, and all staining procedures were carried out on paraffin sections. Collagen was evidenced by staining with: (1) Gomori's trichrome; (2) sirius red, followed by observation under polarized light, and (3) an antihuman collagen type-III antibody. Elastin and the whole elastic system were revealed using an antihuman elastin antibody and Weigert's resorcin fucsin, respectively. RESULTS: At this stage of fetal development, the albuginea is formed predominantly by dense bundles of collagen. Near the corpora cavernosa, the presence of type-III collagen was also observed. Weigert staining showed numerous fibers of the elastic system in the albuginea. Type-III collagen was found to be strongly positive in the cavernous trabeculae and in the connective sheath surrounding the central artery. Using Weigert staining and an immunolabeling method with primary antibody against human elastin, we found an important quantity of elastic system fibers in the trabeculae of the corpora cavernosa. CONCLUSION: In fetuses at 28 WPC the albuginea is formed predominantly by dense bundles of collagen. The trabecular structures of the corpora cavernosa present a significant quantity of type-III collagen and elastic system fibers.

Distribution of elastic system fibres in human fetal liver.

Elastic system fibres are extracellular matrix components found in different organs for which they provide elasticity and some mechanical resistance. Oxytalan, elaunin and elastic fibres, which possess graduated amounts of elastin, are the 3 forms of elastic system fibres that are identifiable by their tinctorial and ultrastructural features. The distribution of these fibres in adult human liver is well-established but little, if anything, is known about them in fetal liver. The distribution of elastic system fibres was therefore investigated in human fetal liver, and the process of elastogenesis characterised. Specimens of liver from 24 human fetuses ranging in age from 13 to 38 wk postfertilisation were studied. The results are presented in relation to gestational age and the size of the portal tracts. Portal tracts exhibited a network of oxytalan fibres at 13 wk; elaunin fibres appeared later after 20 wk postfertilisation. Elastogenesis occurred more rapidly in venous than in arterial walls, and in veins it was more evident in the adventitia. A microfibrillar network of oxytalan fibres was observed around biliary ducts from the outset of their development. Elastogenesis follows the sequence oxytalan, elaunin and elastic fibres, but the elastogenetic process only completes its maturation in arterial walls, thus leading to the internal elastic lamina.