Translational Research in Pediatric Urology: Basic and Clinical Aspects [1st ed.] 9783030502195, 9783030502201

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Translational Research in Pediatric Urology Basic and Clinical Aspects Luciano Alves Favorito Editor

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Translational Research in Pediatric Urology

Luciano Alves Favorito Editor

Translational Research in Pediatric Urology Basic and Clinical Aspects

Editor Luciano Alves Favorito Urogenital Research Unit - Department of Anatomy Rio de Janeiro State University Rio de Janeiro Rio de Janeiro Brazil

ISBN 978-3-030-50219-5    ISBN 978-3-030-50220-1 (eBook) https://doi.org/10.1007/978-3-030-50220-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Translational research implements a “bench-to-bedside,” from laboratory experiments through clinical trials to point-of-care patient applications. The end point of translational research is the production of a promising new treatment that can be used with practical application which can then be used clinically. The present book Translational Research in Pediatric Urology: Basic and Clinical Aspects is a work of the Urogenital Research Unit that was founded by Prof. Francisco J.B. Sampaio 30 years ago. The Urogenital Research Unit, located at the Biomedical Center of the State University of Rio de Janeiro, Brazil, and under the direction of Francisco J.B. Sampaio, is dedicated to advancing the knowledge on the urogenital system, from both a basic science and a medical perspective. Research is conducted on several models, which include clinical trials and experiments on laboratory animals, and employs a wide range of methodologies. In this book, we present basic studies about the urogenital system with anatomic and embryologic emphasis applied to pediatric urology. This book applies to pediatric surgeons, urologists, PhD researchers, fellows, and medical students, and we hope that they will find it useful to clinical practice and medical research. Rio de Janeiro, Rio de Janeiro, Brazil

Luciano Alves Favorito, MD, PhD

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Acknowledgments

I would like to thank the National Council for Scientific and Technological Development (CNPq–Brazil), Coordination of Improvement of Higher Education Personnel (CAPES-Brazil), and the Rio de Janeiro State Research Foundation (FAPERJ) for the Urogenital Research Unit Support.

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Contents

1 Basic Embryology of Urogenital System��������������������������������������������������   1 Luciano Alves Favorito 2 Basic Anatomy of Urinary Tract��������������������������������������������������������������  17 Francisco Jose B. Sampaio and Luciano Alves Favorito 3 Basic Anatomy of the Male Genital System��������������������������������������������  27 Luciano Alves Favorito and Francisco Jose B. Sampaio 4 Basic Anatomy of the Female Genital Tract��������������������������������������������  49 Luciano Alves Favorito 5 Testicular Migration����������������������������������������������������������������������������������  59 Luciano Alves Favorito 6 Basic Research Applied to Undescended Testis ��������������������������������������  77 Luciano Alves Favorito 7 Basic Research Applied to Testicular Torsion ����������������������������������������  99 Luciano Alves Favorito, Diogo B. de Souza, Daniel Hampl, Carina T. Ribeiro, Marco A. Pereira-Sampaio, and Francisco Jose B. Sampaio 8 Methods of Basic Research Applied to Urinary and Genital Systems During the Human Fetal Period �������������������������� 119 Luciano Alves Favorito and Francisco Jose B. Sampaio 9 Basic Research Applied to Hypospadias�������������������������������������������������� 135 Luciano Alves Favorito 10 Basic Research Applied to Renal Anomalies ������������������������������������������ 153 Luciano Alves Favorito, Andre L. Diniz, and Francisco Jose B. Sampaio 11 Basic Research Applied to Ureteral Anomalies �������������������������������������� 169 Luciano Alves Favorito ix

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Contents

12 Basic Research Applied to Bladder Anomalies���������������������������������������� 181 Luciano Alves Favorito, Waldemar S. Costa, and Francisco Jose B. Sampaio 13 Basic Research Applied to Female Reproductive Tract�������������������������� 193 Luciano Alves Favorito and Rodrigo R. Vieiralves 14 Neural Tube Defects and Genitourinary System������������������������������������ 201 Luciano Alves Favorito, Rodrigo R. Vieiralves, Rodrigo S. Pires, and Francisco Jose B. Sampaio 15 Prune Belly Syndrome and Urogenital System �������������������������������������� 219 Luciano Alves Favorito, Carla Mano Gallo, and Francisco Jose B. Sampaio Index�������������������������������������������������������������������������������������������������������������������� 233

Contributors

Waldemar  S.  Costa, BSc  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Andre  L.  Diniz, MD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Luciano  Alves  Favorito, MD, PhD  Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil Carla  Mano  Gallo, BSc  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Daniel Hampl, MD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Marco A. Pereira-Sampaio, MSc, PhD  Department of Morphology, Fluminense Federal University, Niterói, RJ, Brazil Rodrigo  S.  Pires, MD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Carina  T.  Ribeiro, BSc  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil Francisco  Jose  B.  Sampaio, MD, PhD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq –Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil

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Contributors

Diogo  B.  de  Souza, MSc, PhD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq –Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil Rodrigo R. Vieiralves, MD  Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil

Chapter 1

Basic Embryology of Urogenital System Luciano Alves Favorito

1.1  Development of Urinary System 1.1.1  Kidney The urinary system develops from the intermediate mesoderm that extends over the dorsal wall of the embryo (Sadler 1995; Moore 1977; Maizels 1992). During intrauterine life, man develops three kidneys that arise in the following order: pronephros, mesonephros, and metanephros (definitive kidney). Pronephros develops at the third week of pregnancy and suffers apoptotic degeneration and disappears until the fifth week of pregnancy (Pole et  al. 2002). Mesonephros arises on the medium region of the embryo, around the fourth week of pregnancy, giving rise to tubular structures. Until the fourth month, mesonephros disappears completely, except for a few elements that persist at maturity. At the mesenchyme located at the lateral region of the developing mesonephros, mesonephric ducts emerge and progress caudally and merge with the terminal portion of the primitive cloaca (Thomas et al. 2002). Canalization of mesonephric ducts forms the excretory unit with transient function (Fig.  1.1). Mesonephric vesicles and tubules are formed cranio-caudally in all thoracic-lumbar direction. Cranial pairs degenerate coincidentally with the development of caudal pairs and definitive mesonephros at this moment contain around 20 pairs confined at the first three lumbar segments. Definitive kidney or metanephros is formed at the sacral region of the embryo, from a pair of new structures called ureteral bud or metanephric diverticulum that arise from the distal portion of the mesonephric duct. Therefore, the ureteral bud starts from a projection of the mesonephric duct close to its entrance at the cloaca. L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_1

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Fig. 1.1  Schematic drawing showing the development of the pronephros (dotted); mesonephros (dark area), right figure; and metanephrogenic blastema (bigger arrow) and ureteral bud (smaller arrow), left figure

In the beginning of the fifth week of development, the ureteral buds originate at the distal portion of the mesonephric ducts and progress to the more caudal portion of the intraembryonic blocks, the metanephros (Thomas et al. 2002; Glassberg 2002). Fusion of the ureteral bud with the metanephrogenic blastema around the 32nd day of development begins the nephrogenic process (Glassberg 2002) (Fig. 1.1). The ureteral bud stimulates the mesenchymal-epithelial transformation that will originate the metanephric kidney. This transformation does not occur if the ureteral bud is absent. It is speculated that the molecules produced by the ureteral bud induce the modifications that occur at the metanephrogenic blastema (Glassberg 2002). The bifurcation of the ureteral buds will determine the pyelocaliceal pattern and the corresponding renal lobules (Maizels 1992). Nephrons are developed from the eighth week of pregnancy and between the 32nd and 36th weeks the ramifications of the ureteral buds are finished and meet the renal units; although macroscopically formed, they will continue to mature following birth (Short and Smyth 2016). Between the 6th and 10th weeks, the embryonic kidneys will ascend through the posterior abdominal wall until they reach their definitive position at the lumbar region (Fig.  1.2). The exact mechanism by which this migration occurs is not known, but it is postulated that occurs due to differential growth of the sacral and lumbar regions of the embryo (Maizels 1992). During this ascent, the kidneys modify the origin of their vascularization, until definitive arteries are formed in the lumbar region of the embryo. Several anomalies may occur during the renal embryogenic process: agenesis, dysplasia, cystic anomalies, rotation anomalies, vascular anomalies, ascent

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Fig. 1.2 Schematic drawing showing the renal ascent (arrows) that occurs between the sixth and tenth weeks of development

anomalies, and fusion anomalies. Ascent and fusion anomalies are the most frequent (Sadler 1995; Moore 1977; Maizels 1992) and are shown in Fig. 1.3.

1.1.2  Ureter Kidney and ureter collecting systems arise at the ureteral bud, as a dorsal invagination of the mesonephric duct, around the 28th day after conception (Sadler 1995; Moore 1977; Maizels 1992). The ureteral bud has a superior portion that is dilated and a tapered inferior portion. The dilated portion origins the renal pelvis and the tapered inferior region, the ureter (Moore 1977). Since the beginning of the development until the 35th day after conception, the ureter is patent along all its extension (Maizels 1992). However, between the 37th and 40th day after conception, the ureteral lumen closes, and only its median portion remains patent (Maizels 1992) (Fig. 1.4). After the 40th day, the ureteral lumen rapidly extends cranial and caudally and again the ureter is patent (Fig. 1.4). Anomalies of the development of the ureter occurs in around 10% of urological patients (Motola et al. 1988). The most common anomalies include partial and total duplication, ectopic orifices, ureterocele, ureterovesical junction incompetence causing ureteral reflux, and intrinsic obstruction of ureter. Figure 1.5 shows a fetus in the second pregnancy trimester with complete duplication of ureter. Congenital ureteral obstructions are most frequent at the pyelo-ureteral and ureterovesical junctions. According to Alcaraz et al. (1991), congenital ureter obstructions may be divided in two major groups: intrinsic ureteral obstructions and

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obstructions of the ureterovesical junction. The last occur due to persistence of the ureterovesical membrane (Chwalla membrane) that temporarily obstruct this region between the 37th and 43th days after conception (Domenech et al. 1973). At 8 weeks after conception, the ureter is a patent tube, however, without smooth muscle fibers (Maizels 1992). Ureter muscular layer starts to form after the passage of urine by this structure, stimulating myogenesis, around 12th week after conception (Maizels 1992).

a

Fig. 1.3 (a) Schematic drawing showing the ascent and fusion of the kidney: (a) horseshoe kidney, (b) pelvic kidney, and (c) crossed renal ectopia with fusion. (b) Third gestational trimester fetus with horseshoe kidney. (c) Second gestational trimester fetus with renal ascent anomaly – pelvic kidney. (d) Excretory pyelography of a 12-year-old female patient with crossed renal ectopy and fusion

1  Basic Embryology of Urogenital System Fig. 1.3 (continued)

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Fig. 1.4  Schematic drawing showing obliteration and recanalization of ureter during embryonic development. (a) Ureteral bud arising from the mesonephric duct, 28 days after conception. (b) Between days 37 and 40 after conception, ureteral lumen is progressively obstructed. (c) Ureteral lumen is again patent, beginning at the medium portion. (d) After the 40th day of conception, ureteral lumen is again patent in all its extension

1.1.3  Bladder Primitive cloaca is divided by the uro-rectum septum, from the fourth to the seventh week after conception (Sadler 1995; Moore 1977; Maizels 1992). Cloaca is divided in two parts: anus-rectum canal (posterior) and urogenital sinus (anterior) (Fig. 1.6). Cloacal membrane is also divided in two parts: anteriorly, the urogenital membrane and, posteriorly, the anal membrane. The urogenital sinus that arises from the primitive cloaca is divided in three parts: vesical, pelvic, and phallic. The vesical part is the more superior and larger part of the urogenital sinus. Initially, it is continuous to the allantoid, whose lumen closes posteriorly giving rise to the urachus (Sadler 1995; Moore 1977; Maizels 1992). The second part of the urogenital sinus, below the vesical portion, is the pelvic part that will give rise to the prostate and membranous part of the urethra (Fig. 1.6). The distal part of the urogenital sinus is the phallic portion that is externally closed by the urogenital membrane (Fig. 1.6).

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Fig. 1.5 Second gestational trimester fetus showing complete duplication of left ureter

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Fig. 1.6  Schematic drawing showing the division of the cloaca that will give rise to the urogenital sinus, between the fourth and sixth week of pregnancy. (a) Primitive cloaca. (b) Division of cloaca by the uro-rectal septum (arrows); (c) finishing of the division of the cloaca, forming the anterior urogenital sinus and the posterior anus-­rectum canal

Around the fifth week of development, the distal part of the mesonephric duct in relation the ureteric buds dilates and is absorbed by the urogenital sinus region (Maizels 1992). The mesonephric ducts merge at the median line and origin a triangular region, the future bladder trigone (Park 2002).

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Then, the bladder is divided embryologically in two portions: body and trigone. The bladder body derives from the endoderm of the vesical region of the urogenital sinus. The epithelium of this region is derived from the urogenital sinus endoderm; and lamina propria, muscular layers, and adventitia derive from the adjacent splanchnic mesenchyme (Sadler 1995; Moore 1977; Maizels 1992). Bladder trigone origins with the incorporation of the mesonephric ducts at the base of the developing bladder (Park 2002). Initially, these ducts contribute to form the bladder trigone mucosa. However, this epithelium is replaced by the endodermic epithelium of the urogenital sinus (Sadler 1995; Moore 1977; Maizels 1992). Several congenital anomalies may affect the bladder, and the main include: urachus anomalies (due to fail of the allantoid closure), septum anomalies, bladder congenital diverticula, and bladder exstrophy.

1.2  Development of the Male Genital System 1.2.1  Testicle The human testicle origins from a thickening of the coelomic epithelium at the medial region of the mesonephric duct. During the embryonic development, at the second pregnancy trimester, the testicle goes through the abdominal wall, passes by the inguinal canal, and reaches the scrotum (Fig. 1.7). This migration begins around the 24th week after conception and completes around the 32nd week (Backhouse 1982; Heyns and Hutson 1995). Early studies showed that all fetuses older than 30  weeks after conception already showed testicles at the scrotum (Fig.  1.7) (Sampaio and Favorito 1998). The first indication of gonadal development occurs at the fifth pregnancy week, with the thickening of the coelomic epithelium at the medial region of the mesonephros, the genital crest. At this location, epithelium chords (primary sexual chords) grow to the interior of the subjacent mesenchyme, beginning to form the undifferentiated gonad (Moore 1977). At the sixth week of pregnancy, the primitive gonad is composed by a superficial germinative epithelium and by an intern blastema. At the seventh week, the testicular differentiation factor or SRY gene, located at the short arm of the Y chromosome, determines the development of the medullar layer of the undifferentiated gonad, turning it into the testicle (Rosansky and Bloom 1996). The primary chords condense, branch, and anastomose, in a radial and convergent way toward the mesorchium, forming the seminiferous tubules, rectum tubules, and later the rete testis. Right after, they lose their connections with the superficial epithelium, due to the development of a thick and fibrous capsule, called tunica albuginea (Moore 1977).

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Fig. 1.7  Scheme showing the chronology of the testicular migration. (a) Fetuses with 10–20 weeks of pregnancy had the majority of testicles located at the abdomen; (b) fetuses with 21–25 weeks of pregnancy had the majority of testicles in the inguinal canal, and (c) all fetuses with more than 30 weeks of pregnancy had the testicles located at the scrotum

Primordial germ cells migrate by ameboid movements from the caudal portion of the yolk sac to the genital crest. This migration completes at the end of the sixth week of pregnancy (Lamb 1993). Later, they differentiate in gonocytes, penetrating the sexual chords that will form the seminiferous tubules. Some gonocytes adhere to the basal membrane of the tubules and differentiate in fetal spermatogonia (Kogan et al. 1996). Sertoli cells develop at the sixth and seventh weeks, from the coating epithelium. They produce the inhibitor Muller factor that provokes the regression of the paramesonephric duct (Muller duct) (Rosansky and Bloom 1996). At the male embryo, the superior ends of the paramesonephric ducts are responsible for the formation of the Morgagni sessile hydatid, identified at the superior poles of the testicles and currently called testicular appendices (Noske et al. 1998).

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Around the ninth week of pregnancy, the Leydig cells, derived from the mesenchyme, produce testosterone, stimulating the formation of the mesonephric duct (Wolff duct) at the male genital tract (Backhouse 1982; Rosansky and Bloom 1996). When the mesonephros degenerates, around 5–12 mesonephric tubules close to the differentiating testicle (epigenital tubules) persist, lose their primitive glomerulus, and fuse with the rete testis, forming the efferent tubules (Sadler 1995, 18). This fusion occurs around the 12th week of pregnancy, coincident to the beginning of the canalization of the rete testis and of the mesonephric tubules, that will complete only at puberty (1,2, Rosansky and Bloom 1996). At the third month of fetal life, the testicle is located at the retroperitoneum future pelvic cavity. The testicle develops at the medial portion of the mesonephros and is attached to the future area of the inguinal canal by a mesenchyme column known as testicular gubernaculum (Moore 1977) (Fig. 1.8). During the second gestational trimester, the testicle and epididymis start to migrate from the abdomen to the scrotum, completing this descent around the 30th week of pregnancy (Sampaio and Favorito 1998). Fig. 1.8  Scheme showing the development of the testicle and formation of the epididymis. T testicle, G gubernaculum, DW Wolff duct, SU urogenital sinus, arrow mesonephric ducts

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G

DW

G

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1.2.2  Epididymis and Deferens Duct Epididymis and deferens duct originate from the mesonephric duct. Mesonephric tubules (epigenital ducts), due to the proximity with the differentiating gonads, fuse with the testicle tubules (Fig. 1.8). Usually, 5–12 mesonephric tubules unite with the rete testis, forming the efferent tubules (Scorer and Farrington 1971). The portion of the mesonephric ducts adjacent to the testicle becomes elongated and convolute forming the epididymis. The remaining portion of the mesonephric duct forms the deferens duct. This urogenital fusion occurs around the 12th week after conception. During the fourth week of embryonic development, the efferent tubules adjacent to the testicle remain straight, while the tubules adjacent to the deferens duct hank (Maizels 1992). Therefore, the epididymis has a double embryonic origin: the head originates from the genital margin, and the body and tail origin from the superior portion of the hank deferens duct. There may be congenital anomalies at the region of the efferent tubules, causing disjunction of the testicle and epididymis (Barthold and Redman 1996). There are anatomic anomalies where it is observed agenesis of the body and tail of the epididymis, while the head is positioned at the correct place (Hinman 1993). Disjunction anomalies (Fig.  1.9) or atresia may be explained by the double embryonic origin of the epididymis or by vasculature alterations of the mesonephric duct that may occur in this period (Barthold and Redman 1996). These anomalies are frequently associated to cryptorchidism (20) and are also observed in infertile patients. In males without cryptorchidism and in human fetuses without congenital anomalies (Scorer and Farrington 1971), the incidence of epididymal anomalies is rare (less than 5%). a

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Fig. 1.9  Disjunction anomalies of the epididymis. (a) Disjunction of the epididymal tail, (b) total disjunction of epididymis and testicle, and (c) total disjunction of epididymis and testicle, with the epididymis located more caudally than the testicle

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1.2.3  Prostate and Seminal Vesicles Prostatic urethral epithelium close to the orifices of the ejaculatory ducts derives from the endoderm of the vesical part of the urogenital sinus. The remaining of the prostatic urethra epithelium derives from the pelvic part of the urogenital sinus. The primitive prostatic ducts develop at the prostatic urethra under the influence of the mesenchyme of the mesonephric duct (Wolff) (Fig. 1.10). Fetal androgens start to be produced by the testicle during the eight week and are required for the inductive activity of the mesenchyme. Between the 11th and 12th gestational weeks the mesenchyme around the prostatic urethra is stimulated by androgens inducing the proliferation of the epithelium. Initially, the ducts are solid and after the 30th week they acquire a lumen. In the beginning, small collections of cellular buds are developed and posteriorly acinar structures arise (Fig. 1.11). Finally, as the ducts invade the surrounding mesenchyme, lobular groups of acinar-tubular structures are developed. Prostatic ducts arise from three areas of the epithelium and from the contiguous mesenchyme, at the portion of the urogenital sinus, that will form the floor of the prostatic urethra (Sadler 1995; Moore 1977; Maizels 1992). Each of the three groups of ducts will drain each of the three zones of the prostate. The first group of ducts sprouts distally to the seminal colliculus and forms the peripheral zone of the prostate. The second group sprouts at the urethra in two rows next and above the exit part of the ejaculatory ducts and forms the central zone. The third group, located at the vesical-urethral region, will proliferate in the interior of the deep mucosa forming the ducts and glands of the transition region (Baskin 2000).

Ducto de müller Ducto de wolff

Ureter

Ubérculo de wolff tubérculo de müller

Fig. 1.10  Schematic drawing of the development of the prostatic ducts from the influence of the mesenchyme of the Wolff duct. Ducto de Muller – Muller duct; ducto de Wolff – Wolff duct; tuberculo de Wolff – Wolff tubercle; tuberculo de Muller – Muller tubercle

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Bexiga

Ducto ejaculador

Ureter Próstata Vesícula seminal

Uretra

Fig. 1.11  Schematic drawing of the development of the prostate, in a more advanced phase, with the beginning of the development of the acinar structures. Bexiga – bladder; prostate – prostate; ducto ejaculatorio – ejaculatory duct; vesicular seminal – seminal vesicle; uretra – urethra

1.2.4  Penis The knowledge of the embryology of the penis eases the understanding of several congenital anomalies such as hypospadias, epispadias, and phimosis. Detailed description of penile embryology will be presented at Chap. 9.

1.3  Development of the Female Genital System 1.3.1  Ovary The ovaries develop at the abdominal cavity, in the medial region of the mesonephric duct, from a thickening of the coelomic epithelium, located in that region, around the seventh week of development (Sadler 1995; Moore 1977; Maizels 1992). The ovary also migrates; however, the presence of the uterus prevents the displacement outside the abdominal cavity, causing the ovary to locate at the pelvic region (Park 2002). As in males, there is a mesenchymal structure attached to the lower pole of the ovary. This structure unites the lower pole of the ovary to the region of the large

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vaginal lips, and it is called female gubernaculum or round ligament that will be a part of the female inguinal canal (Park 2002).

1.3.2  Uterus, Fallopian Tube, and Vagina The uterus and the uterine tubes origin from the paramesonephric ducts between the tenth and 20th week of pregnancy (Thomas et al. 2002). These ducts present three portions: (a) cranial portion that opens to the coelomic cavity, (b) horizontal portion that crosses the mesonephric duct, and (c) caudal portion that fuses to its homologous counterpart (Sadler 1995; Thomas et al. 2002) (Fig. 1.12). With the migration of the ovary, both first portions will give rise to the uterine tube, and the fused distal portion will form the uterine canal (Sadler 1995) (Fig. 1.12). In the absence of testosterone, the mesonephric ducts degenerate, forming few remnants including the epoophoron and paroophoron, located close to the uterine tube, and the Gartner cysts, close to the superior portion of the vagina (Sadler 1995). a

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Fig. 1.12 (a) Development of the female genital tract. (a) Fusion of the paramesonephric ducts (bigger arrow) with the sinus-vaginal bulbus (smaller arrow), (b) formation of the vagina, and (c) final aspect of female genitalia after the end of canalization of uterus and vagina. (b) Photograph of a 22-week pregnancy fetus showing formed uterus and attachments

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In the region of the union of the paramesonephric ducts with the urogenital sinus, a tissue condensation is formed, called sinus-vaginal bulbus. Between the 10th and 20th weeks, the sinus-vaginal bulbus will be developed toward the fetal perineum, separating the developing vagina from the urethra. During this period, the canalization of the vagina is observed. The vaginal lumen will remain separated from the urogenital sinus, by a thin tissue called hymen that presents an epithelial layer derived from the urogenital sinus and a thin layer of vaginal cells. The two proximal thirds of the vagina derive from the paramesonephric ducts, while the distal portion origins at the urogenital sinus (Fig.  1.12). The vaginal entrance and the external genitalia derives from the ectoderm (Sadler 1995; Thomas et al. 2002).

1.3.3  External Genitalia In the beginning of the development, the external genitalia is undifferentiated (Parker 2016). Female external genitalia develops due to hormonal stimuli between the third and fifth pregnancy months (Sadler 1995). The genital tuberculum elongates giving rise to the clitoris; the urethral crimps will not fuse giving rise to the minor lips. Genital folds elongate and give rise to the major vaginal lips; the urogenital groove remains open and forms the vaginal vestibule. Figure 1.13 shows the origin of the undifferentiated external genitalia in both sexes (Fig. 1.13).

Fig. 1.13  Undifferentiated genitalia give rise to external male genitalia (left) and female genitalia (right)

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References Alcaraz A, Vinaixa F, Tejedo-Mateu A, Forés MM, Gotzens V, Mestres CA, Oliveira J, Carretero P.  Obstruction and recanalization of the ureter during embryonic development. J Urol. 1991;145:410–6. Backhouse KM.  Embryology of testicular descent and maldescent. Urol Clin North Am. 1982;9:315–25. Barthold JS, Redman JR. Association of epididymal anomalies with patent processus vaginalis in hernia, hydrocele and cryptorchidism. J Urol. 1996;156:2054–6. Baskin LS.  Hipospadias. Anatomy embriology and reconstrutive techniques. Braz J Urol. 2000;26:621–9. Domenech G, Tejedo A, Vilanova J. Contribuition al estudio de la membrana ureteral de Chwalla. Su importancia clinica. Arch Esp Urol. 1973;26:355–9. Glassberg KI. Normal and abnormal development of the kidney: a clinician’s interpretation of current knowledge. J Urol. 2002;167:2339–51. Heyns CF, Hutson JM. Historical review of theories on testicular descent. J Urol. 1995;153:754–67. Hinman JRF. Penis and male urethra. In: Atlas of urological anatomy. Philadelphia: W.B. Saunders Co.; 1993. p. 418. Kogan S, Hadziselimovic F, Howards SS, Snyder IIIHM, Huff D. Pediatric andrology. In: Adult and pediatric urology. 3rd ed. St Louis: Mosby Year Book; 1996. Lamb DJ. Growth factors and testicular development. J Urol. 1993;150:583–5. Maizels M. Normal development of the urinary tract. In: Campbell’s urology, vol. 6a. New York: Saunders; 1992. p. 1301. Moore KL.  The developing human. Clinically oriented embryology. Philadelphia: W.B. Saunders; 1977. Motola JA, Shalon RS, Smith AD. Anatomy of the ureter. Urol Clin North Am. 1988;15:295–8. Noske HD, Kraus SW, Altinkilic BM, Weidner W. Historical milestones regarding torsion of the scrotal organs. J Urol. 1998;159:13–6. Park JM. Normal and anomalous development of the urogenital system. In: Campbell’s urology, vol. 8a. New York: Saunders; 2002. p. 1737. Parker J. Embryol genitourin tract. In: Campbell-Walsh urology. 11th ed; 2016. p. 2824–33. Pole RJ, Qi BQ, Beasley SW.  Patterns of apoptosis during degeneration of the pronephros and mesonephros. J Urol. 2002;167:269–71. Rosansky TA, Bloom DA. The undescended testis: theory and management. Urol Clin North Am. 1996;22:107–18. Sadler TW. Langman’s medical embryology. 7th ed. Baltimore: Williams & Wilkins; 1995. Sampaio FJB, Favorito LA. Analysis of testicular migration during the fetal period in humans. J Urol. 1998;159:540–2. Scorer CG, Farrington GH. Development and descent of the testis. In: Congenital deformities of the testis and epididymis. New York: Appleton-Century-Crofts; 1971. p. 1–27. Short KM, Smyth IM. The contribution of branching morphogenesis to kidney development and disease. Nat Rev Nephrol. 2016;12(12):754–67. Thomas FMD, Rickwood AMK, Duffy PG.  Essentials of paediatric urology. London: Martin Dunitz; 2002.

Chapter 2

Basic Anatomy of Urinary Tract Francisco Jose B. Sampaio and Luciano Alves Favorito

2.1  Kidney Anatomy Kidneys are paired organs located at the retroperitoneum close to the posterior wall of abdomen. They have a unique shape, with an upper pole (superior end) and a lower pole (inferior end), with a convex lateral border and a median concave border. The median border presents a depression, the kidney hilum, with the renal vessels, and renal pelvis or basinet (Testut 1921).

2.1.1  Kidney Position The kidneys are located at the posterior abdominal wall, in contact with the major psoas muscle in each side, and therefore their longitudinal axis parallels the oblique direction of the psoas (Fig. 2.1). The major psoas is cone shaped, therefore the kidneys are also bended posteriorly to the longitudinal axis, and the upper poles are more medially and inferiorly located than the lower poles (Fig. 2.1). The renal hilum

F. J. B. Sampaio Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq –Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil e-mail: [email protected] L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_2

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is anteriorly rotated over the psoas, positioning the kidney borders dorsally. Therefore, the kidneys are angulated at 30–50° posteriorly to the frontal plane (Williams et al. 1995).

2.1.2  Renal Relationships The surface of the kidneys is covered by a fibrous tissue, called renal capsule (real renal capsule). Each kidney is involved by a mass of adipose tissue (perirenal fat) located between the peritoneum and the posterior abdominal wall (Fig. 2.2). The perirenal fat is enveloped by the renal fascia (Gerota’s fascia). The renal fascia is circled anteriorly and posteriorly by another layer of fat tissue, with various thickness, called pararenal fat. The anterior and posterior layers of renal fascia (Gerota’s fascia) subdivide the retroperitoneal space in three potential compartments: Fig. 2.1  Superior view of a transverse section of the kidneys at the level of the second lumbar vertebra in a frozen corpse. The kidneys are angulated between 30° and 50° posteriorly to the frontal (coronal) plane. RK right kidney, LK left kidney, A Aorta artery, P pancreas, D duodenum, and C right colon

Fig. 2.2  Renal wraps: photography of a fresh male fetus of the third trimester of pregnancy showing the position of the kidney in the retroperitoneum. It can be observed the renal fascia and the renal fat by transparency

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(a) The posterior pararenal space that contains only fat (b) The perirenal space that contains the adrenal glands, the kidneys, and proximal ureters, along with the perirenal fat (c) The anterior pararenal space that, in contrast with the intermediate and posterior spaces, is located on both sides of the median line in the abdomen and contains the ascending and descending colons, the second portion of the duodenum, and the pancreas (Fig. 2.3) (Sampaio 1996) Inferiorly, the layers of renal fascial weakly fuse around the ureter (Fig. 2.4a). Superiorly, both renal fascia layers fuse above the adrenal gland and end united to the infradiaphragmatic fascia (Fig. 2.4b). An additional layer of fascia separates the adrenal gland and the kidney (Sampaio 1996). Fig. 2.3  Superior view of a transverse section of the kidneys at the level of the second lumbar vertebra. The three compartments of the retroperitoneal space are indicated. P pararenal space, I intermediate perirenal space, A anterior pararenal space

a

A I P

b

Fig. 2.4  Anterior view of the kidneys and renal fascial (Gerota’s fascia). (a) Anterior view of the lower pole of right kidney (RK) showing the relationship between renal fascia (RF) and the psoas muscle. (b) Anterior view of the relation between the right kidney (RK) and the liver in a frozen corpse, showing the relationship between the liver and the right kidney and the hepatorenal space (*)

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Laterally, both layers of the renal fascia fuse behind the descending and ascending colon. Medially, the posterior fascial layer fuses to that of the spinal muscles. The anterior fascial layer mixes with the connective tissue of the great vessels (aorta and inferior vena cava).

2.1.3  Parietal Relations of the Kidneys The kidneys rest over the major psoas and square lumbar muscle. Usually, the left kidney is higher than the right one, and the posterior surface of the right kidney is crossed by the 12th rib and the posterior surface of the left kidney is crossed by the 11th and 12th ribs. The posterior surface of the diaphragm is united to the ends of the 11th and 12th ribs (Fig. 2.5). Close to the vertebral spine, the diaphragm is connected to the muscles of the posterior wall of the abdomen, forming the arched medial and lateral ligaments in each side (Fig. 2.5). Therefore, the posterior surface of the diaphragm is arched as a dome over the superior pole of both kidneys (Sampaio 1996).

2.1.4  Visceral Relations of the Kidneys The kidney maintains relations with the liver, spleen, and colons. Kidney relation with the liver is very important in urology and radiology. The space between the right kidney and liver is called hepatorenal space (Morrison space) that is well visualized in image exams, particularly in the presence of free liquid in the abdominal cavity (Fig. 2.6). Due to their dimensions, the liver and spleen may be positioned posterior and laterally at the level of the supra-hilar region of the kidney.

Fig. 2.5  Inferior view of a transversal section at the level of the first lumbar vertebra of a frozen corpse showing the relation between the left kidney (LK) and the posterior muscles (PM); S spleen

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Fig. 2.6  Superior view of a transversal section at the level of the first lumbar vertebra of a frozen corpse showing the relation between the right kidney (RK) and the liver and the hepatorenal space (*)

Therefore, in endourology, it is important to observe that the space for a high renal puncture is very limited. If the puncture is performed while the patient is medium or intensively breathing, there is a high risk of lesion of the liver or spleen. This knowledge is particularly important in patients with hepatomegaly or splenomegaly (Sampaio 1996). In those cases, it is important to obtain previously a computer tomography before puncturing the kidney. The descending colon extends from the ileum-colic valve until the right colonic flexure (hepatic flexure), becoming the transverse colon. The hepatic colonic flexure (hepatic angle) leans anteriorly to the inferior portion of the right kidney. The descending colon extends inferiorly to the left colonic flexure (splenic flexure) at the level of the iliac crest. The left colonic flexure leans anteriorly and laterally to the left kidney. It is important to consider the retroperitoneal position of the ascending and descending colons. During routine computed tomography (CT) exams it was observed the retroperitoneal colon leaning in a posterior and lateral or retro-renal position. In those cases, there is an increased risk of lesion during a percutaneous intrarenal access. The retro-renal colon is more frequently related to the lower kidney poles (Williams et al. 1995). In a study controlled by CT, the retro-renal colon was observed in 1.9% of cases, while the patients were in a supine position. However, when in a ventral position (more frequent position used for percutaneous renal access), the retro-renal colon was observed in 10% of patients (Hooper et al. 1987). Therefore, in all renal puncture procedures, it is important to carefully analyze the fluoroscopic exam with the patient in ventral decubitus, in order to detect the presence of a retro-renal colon. The fluoroscopic exams are particularly important in the inferior polar region of the kidneys.

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Fig. 2.7  Renal arteries. The figure shows an endocast (Resapol resin) of the abdominal aorta artery originating one renal artery to each kidney

2.1.5  Vascular Anatomy The renal artery is a branch of the lateral aspect of the abdominal aorta that origins around 1.5 cm below the superior mesenteric artery. The right renal artery reaches the renal hilum and runs posteriorly to the inferior vena cava. The renal left artery is shorter than the right one. There are many frequent variations of the main renal artery. It is more common to observe multiple renal arteries than renal veins. The main renal artery is also more prevalent than any other arteries with the same caliber. In a great series of renal pedicles analyzed, it was observed variations of the renal artery in 30% of patients (Sampaio and Favorito 1993; Sampaio 1993) (Fig. 2.7). The main renal artery divides into an anterior and a posterior branch, after originating the inferior adrenal artery. The posterior branch (retro-pyelic artery) continues as a posterior segment artery that irrigates the posterior homonym segment, without significant ramifications, and the anterior branch of the renal artery divides into three or four segment arteries. Before entering the renal parenchyma, the segment arteries divide into interlobar arteries (infundibular arteries) that run adjacent to the calix infundibulum and the smaller calix, reaching the renal columns between the renal pyramids (Sampaio 1996). As the interlobar arteries progress, close to the base of the pyramids, they originate the arched arteries (usually by dichotomy). The arched arteries originate the interlobular arteries that run peripherally forming the glomerular afferent arterioles (Sampaio 1996; Sampaio and Favorito 1993). On the other side, the intrarenal veins do not present a segment pattern. Also, contrary to the arteries, there are free communications along the venous system, with broad anastomosis among veins. Therefore, these anastomoses prevent congesting and ischemia of the renal parenchyma in the presence of venous lesions. The right renal vein is shorter and usually without branches, while the left renal vein is longer, running posteriorly to the aorta, usually receiving blood from three tributaries: gonadal, adrenal, and lumbar veins (Fig. 2.8) (Sampaio 1996).

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a

23

b

Fig. 2.8 (a) Photography of a formalized male corpse showing the renal left artery (red), renal left vein (blue), and ureter (yellow). We can observe the gonadal vein, the left adrenal vein, and the abdominal aorta. In the figure, it is also possible to observe the left ureter in yellow, presenting duplicity; (b) a schematic drawing showing the same structures of the (a)

2.1.6  Lymphatic Drainage Intrarenal lymphatics are divided into superficial and deep plexuses. The superficial plexus is located right after the renal capsule and is attached to cortical lymphatic vessels. In pathologic conditions (e.g., pyelonephritis), this plexus may communicate with an extrarenal plexus, located at the perirenal fat, that drains into aortic lymph nodes in the lumbar region (6). The deep plexus is subcortical and also pyramid shaped (located more profoundly). These deep plexuses are perivascular and drain together, running along with the arched and interlobar vessels, converging to the renal hilum. The collecting channels arise at the renal hilum, and, if the renal artery is present, usually the lymphatic vessel will follow that one. There are one to four lymphatic channels that arise on the renal hilum, anteriorly or posteriorly to the renal vein. These lymphatic channels may anastomose and show a plexiform aspect. Usually they are periarterial, forming an anterior plexus, when arising at the ventral surface of the kidney, or a posterior plexus, when arising at the dorsal surface of the kidney. In some cases, the lymphatic channels may directly connect to their lymph nodes, without following the arterial branches (6). The lymphatic channels of the right kidney may divide into posterior, medium, and anterior channels. The posterior lymphatic vessels follow the renal artery, posteriorly to the vena cava. Such lymphatic vessels reach the aortic-lumbar lymph nodes, located right below the origin of the renal artery. Next, they run and end at the lateral cava lymph nodes and follow the right pillar of the diaphragm muscle, to reach the right abdominal lymphatic chain. The anterior lymphatic channels

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that run above the vessels drain into the dorsal lymph nodes or, when medially located, end at the precava lymph nodes. In some occasions, they may cross the vena cava to reach the superior inter-aorta-cava lymph nodes. The medial lymphatics run between the renal vein and artery, reaching the anterior and posterior lymph node groups. The lymphatic vessels of the left kidney may divide into posterior and anterior branches. The posterior channels leave the renal hilum and run dorsally to the renal vessels to reach the lymph nodes of the pillar of the diaphragm muscle. The anterior channels run ventrally to the renal vein and reach the lymph nodes located above or below the origin of the renal artery, draining the superior or lower pole of the kidney, respectively. There are also lymph nodes that origin at the inferior pole of the kidney to reach the lymph nodes located at the origin of the spermatic artery, at the lateral surface of the aorta (Delmas et al. 1989). It is important to stress that in both sides, the lymphatic drainage of the posterior region of the kidney (dorsal surface) reaches directly the lymph nodes of the diaphragmatic pillars, in contact with the hiatus of the splanchnic nerve. From that point on, through the diaphragm, these lymphatic vessels drain into the retro-aortic lymph nodes from T11 to L1 (mediastinal lymph nodes). This detail is fundamental when analyzing the dissemination of metastasis of renal cell carcinoma.

2.2  Anatomy of Adrenal Glands The adrenal (suprarenal) glands are endocrine, yellowish, small glands, weighing 3–5 g. During the fetal period, the adrenal glands are very big, with the size of one-­ third of the kidney (Fig. 2.9). After birth, the cortical region degenerates and the glands shrink. It has two regions: the peripheral cortex, responsible for the production of glucocorticoids and mineralocorticoids, and the central medullar, responsible for the production of adrenaline and noradrenaline (Netter 1978). They are located between the superior and medial face of the anterior surface of the kidney and the diaphragm. The glands are surrounded by the renal fascia and are located in the perirenal space and involved by fat. The right gland is pyramid shaped, and the base is in contact with the kidney, and the posterior surface is in contact with the diaphragm muscle and inferior vena cava. The left adrenal is semilunar shaped and is posteriorly related to the diaphragm and anteriorly to the pancreas, spleen, and stomach (Williams et al. 1995). The adrenal is irrigated by three arteries and drained by only one vein. The knowledge of the vascular anatomy of the adrenals is of great practical importance

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Fig. 2.9  Photography of a fresh male fetus with 20 weeks after conception. The anterior abdominal wall was opened and the liver mobilized. We can observe the right kidney (RK) and the right adrenal gland (A). It is possible to observe that at that gestational age the adrenal gland is big, corresponding to approximately 1/3 of the fetal kidney. RT right testis

during their surgical removal, particularly by laparoscopy. The three arteries that irrigate the gland are superior adrenal (branch of the lower phrenic); medial adrenal, a direct branch of the aorta; and the inferior adrenal, a branch of the renal artery (Fig.  2.10). The arterial distribution is the same in both sides (Williams et al. 1995). There is only one vein that drains into the adrenal, the adrenal vein, which is, at right, an affluent of the inferior vena cava, and, at left, that drains into the left renal vein. The adrenal vein shows a relatively large caliber and is one of the main structures that must be ligated during resection surgeries of the gland (Netter 1978; Williams et al. 1995).

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Fig. 2.10 Schematic drawing showing the arterial irrigation of the adrenal gland. 1, superior adrenal artery; 2, medial adrenal artery; and 3, inferior adrenal artery

References Delmas V, Hidden G, Dauge MCI.  Remarques sur les lymphatiques du rein: le premier relais nodal. Bull Soc Anat. 1989;13:105. Hooper KD, Sherman JL, Luethke JM.  The retrorrenal colon in the supine and prone patient. Radiology. 1987;162:4433. Netter F. Reproductive system, vol. 2. Summit: CIBA; 1978. Sampaio FJB. Renal arterial pedicle: anatomic analysis applied to urologic and radiologic procedures. In: Sampaio FJB, Uflacker R, editors. Renal anatomy applied to urology, endourology and interventional radiology. New York: Thieme Medical; 1993. p. 47. Sampaio FJB. Anatomy of the kidney for endourology. In: Segura JW, editor. Smith’s textbook of endourology. Part II: percutaneous surgery. Quality Medical: St. Louis; 1996. p. 150. Sampaio FJB, Favorito LA. Ureteropelvic junction stenosis: vascular anatomical background for endopyelotomy. J Urol. 1993;150:1787. Testut L.  In: Livre X, editor. Anatomíe humaine: appareil uro-génital. 7th ed. Paris: Librairie Octave Doin; 1921. Williams PL, Bannister LH, Berry MM, Dyson M, Dussek JE, Ferguson MWJ. Gray’s anatomy. 38th ed. Edinburgh: Churchill-Livingstone; 1995.

Chapter 3

Basic Anatomy of the Male Genital System Luciano Alves Favorito and Francisco Jose B. Sampaio

3.1  Prostate Anatomy The prostate is frequently afflicted by pathologies such as prostate cancer, benign prostatic hyperplasia, and prostatitis. Knowledge of the organ’s anatomy is necessary to perform surgical interventions. The prostate has a base, apex, anterior face, posterior face, and two inferolateral faces. The base of the prostate is continuous with the bladder neck and the smooth musculature passes from one organ to the other without interruption. The urethra penetrates the central region of the prostate base. From below, the prostate apex rests on the upper surface of the urogenital diaphragm (Fig. 3.1). The urethra leaves the prostate just above the apex, on the anterior surface. From the front, the anterior surface of the prostate is related with the pubic symphysis, separated from it by the extraperitoneal fat in the retropubic space. From behind, the posterior surface of the prostate is intimately related with the anterior surface of the rectum and separated from this by the retroprostatic septum (Denonvilliers’ fascia) (Brooks 2002).

L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil F. J. B. Sampaio (*) Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq –Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_3

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28 Fig. 3.1 (a) Diagram of a sagittal section of a male pelvis indicating the relations of the prostate. (b) Frontal section of the pelvis showing the prostate relations; 1 pubis, 2 external obturator muscle, 3 periprostatic veins, and 4 ejaculatory ducts

L. A. Favorito and F. J. B. Sampaio

a

b

3.2  Prostate Zonal Anatomy According to the traditional division that is still adopted in the majority of books on urology and anatomy (Lowsley classification), the prostate has six continuous lobes that are not macroscopically or microscopically separable: anterior lobe, posterior lobe, right and left lateral lobes, medial-commissural lobe, and medial-subcervical

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lobe (Albarran’s glands) (Fig.  3.2). However, these lobes only appear as distinct entities in individuals suffering from benign prostatic hyperplasia, and even in these cases, there are several anatomical variants (Sampaio 1992). Various studies by McNeal and colleagues changed the classic concept of the anatomical division of the prostate into lobes, instead indicating that this division exists under normal conditions only during the fetal period (McNeal et al. 1988). In recent years, based on histological differences, it has been described that in adults the glandular tissue of the prostate represents two fused glands in a single structure. These two parts are called the central zone and peripheral zone (Fig. 3.3) (McNeal et al. 1988). The central zone consists of a portion of the glandular tissue surrounding the ejaculatory ducts. It has an apex, located near the seminal colliculus (verumontanum), and a base, located above and behind the bladder neck (Fig. 3.3). The central zone accounts for about 20% of the total mass of the prostatic glandular tissue. The peripheral zone is the largest region of the prostate, making up about 70% to the total glandular mass. It is represented by a pair of ducts that originate in the posterolateral recess of the urethra wall and radiate laterally (Fig. 3.3). These ducts extend in a continuous double line from the prostate apex almost to the bladder neck, and since they are lateral and posterior, they maintain an anatomical relation with the seminal colliculus. Together, the central and peripheral zones account for 90–95% of the prostatic glandular tissue (McNeal et al. 1988). The main anatomical characteristic of the urethra, which is located cranially to the base of the verumontanum, is a cylindrical sphincter muscle that surrounds the urethral submucosa until the region of the bladder neck. Nearly all the ducts located along this urethral segment are encircled by this sphincter, being restricted to the submucosa area. Therefore, the full development of these periurethral glands is constrained by their confinement, so that together they represent less than 1% of the prostatic glandular tissue. Fig. 3.2  Classification of Lowsley. The prostate has six lobes: anterior lobe (a), posterior lobe (p), right and left lateral lobes (lat), medial-commissural lobe (mc), and medial-­ subcervical lobe (Albarran’s glands). U urethra, vd vas deferens

a

u lat

lat

de

mc de post

30 Fig. 3.3 (a) Diagram showing the zonal anatomy of the prostate according to McNeal. Zt transition zone, Zc central zone, Za anterior zone, Zp periferic zone, u urethra, vd vas deferens. (b) Diagram showing only the transition zone. U urethra, vd vas deferens

L. A. Favorito and F. J. B. Sampaio

a

b

The most distal region of the periurethral sphincter contains more complex ducts and an acinar system larger than those described previously. This subregion is known as a transition zone and accounts for between 5% and 10% of the normal glandular tissue (Fig.  3.3). Although small and insignificant from a functional standpoint, the pre-prostatic region (periurethral submucosa glands  +  transition zone) has high anatomical complexity. The sphincter region described previously can be analyzed as an intrusion from the bladder neck in a prostatic glandular region. At this point, two types of tissue compete for space. As a result, the sphincter tissue (originating from the bladder neck) becomes fully developed, while the glandular component remains atrophied (McNeal et al. 1988). This relationship between the tissues of different types and origins can be important to understand benign prostatic pathology. Finally, a considerable region of the prostate, called the anterior zone (anterior fibromuscular stroma), is entirely nonglandular, instead consisting mainly of smooth muscle fibers. The anterior zone does not play an important role in the prostate’s function, but its location and extension increase the difficulty of visualizing and studying the glandular tissue of the anterolateral region of the prostate (McNeal et al. 1988).

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3.3  External Sphincter of the Urethra The segment of the urethra located between the prostate apex and penile bulb is covered by a cylinder with vertical orientation called the external striated urethral sphincter (Oerlich 1980; Brooks et al. 1998). This arrangement of the sphincter is different than the traditional description by which the urogenital diaphragm and the sphincter form a horizontal plane uniting the ischiopubic branches between the upper and lower fascias of the perineum (Netter 1978). In the membranous urethra, this striated muscle forms a concentric ring of circular fibers that is thickest in front, diminishes progressively on the lateral faces, and becomes sparse with semilunar form in the rear (Fig. 3.4). The direction of the fibers changes from transversal (in front) to longitudinal (on the sides). This muscle layer is separated from the urethral mucosa by submucosal glands and smooth muscle tissue (Manley 1966). The majority of these fibers have peripheral arrangement, except in the anterior median line, where some fibers can be seen within the anterior vestigial lobe. These fibers are mixed with the fibromuscular stroma and glands, diminishing toward the urethral lumen (Manley 1966). The transversal fibers that cover the anterior face end abruptly half way between the prostate apex and bladder neck, slightly above the seminal colliculus. In some cases, the striated muscle continues advancing proximally in symmetric form as distinct tracts of primary longitudinal fibers that cover the anterolateral faces of the prostate. The striated muscle fibers near the urethra obliquely cross an arch of striated muscle. The active or tonic contraction of this musculature occurs around the anterior and posterior walls of the urethra, near the seminal colliculus, closing the

Fig. 3.4 Photomicrograph of the prostate of a fetus in the second gestational trimester, indicating the arrangement of the external striated sphincter (1) of the urethra (U) with the anterior region of the prostate. Note that the striated fibers of the sphincter (1) are independent of the smooth musculature; 2 vas deferens (Masson’s trichrome, 40×)

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bladder neck while at the same time closing the distal urethra by the contraction of the circular portion. In cross sections of the prostate apex, the striated muscle is circular, enveloping the entire urethra, and in the middle third it dislocates laterally, while in the bladder neck it is only seen in the posterolateral regions (Oerlich 1980; Brooks et al. 1998). Near the bladder neck, the fibers diminish and eventually disappear, but a few can be seen mixed with the smooth muscle tissue on the lateral face of the vesical trigone. This smooth muscle forms the internal urethral sphincter, because it emerges from the anterior wall of the bladder and gradually diminishes over the anterior face of the prostate. Some of its fibers probably penetrate the pubovesical or puboprostatic ligament (Manley 1966).

3.4  Prostatic Vascularization 3.4.1  Arteries The prostate is irrigated by the inferior vesical artery and medial rectal artery, branching from the anterior trunk of the internal iliac artery. The inferior vesical artery is the most important, and when it approaches the gland it splits into two main branches: urethral artery and capsule artery. The urethral artery penetrates the gland through the prostate-vesical junction, posteriorly, and connects to the bladder neck, being responsible for a large part of the vascularization in cases of benign prostatic hyperplasia. When the adenoma is resected, the main bleeding points are on the bladder neck in the 4 and 8 o’clock positions (Fig. 3.5) (Brooks 2002).

Fig. 3.5 Diagram demonstrating the arterial branches of the prostate (*)

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3.4.2  Periprostatic Venous Plexus The periprostatic venous plexus is derived from the deep dorsal vein of the penis, which passes between the pubic arch and the striated urethral sphincter, entering the pelvis, where it divides into three branches: the central superficial branch and two lateral plexus branches (Fig. 3.6). The superficial branch is located between the two puboprostatic ligaments and drains the retropubic fat, the front wall of the bladder and the rear portion of the prostate. The lateral plexuses are located beside the prostate and receive drainage from the rectum, in communication with the vesical plexus. There are three to five veins that originate from the lateral plexus that drain into the internal iliac vessel. These veins communicate with the emissary veins of the pelvic bones and with the vertebral plexus, thought to be involved in the dissemination of prostate tumors (Brooks 2002). Variations are frequent in the location and distribution of the periprostatic veins (Myers 1991).

3.5  Lymphatic Drainage of the Prostate The lymphatic drainage of the pelvis is performed mainly by the internal iliac lymph nodes. These lymph nodes have three main chains: presacral, obturator, and internal pudendal lymph nodes. The lymphatic drainage of the prostate goes to the internal iliac lymph nodes, principally to obturator nodes (Weingartner et al. 1996). Fig. 3.6 Diagram demonstrating the formation of the periprostatic venous plexus with the anterior and lateral regions of the prostate. 1 superficial branch, 2 deep lateral branches

2 1

2

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3.6  Innervation of the Prostate and Neurovascular Bundle The innervation of the prostate originates from the inferior hypogastric plexus, in turn which originates from the union of the hypogastric nerve (branch of the superior hypogastric plexus) with the pelvic splanchnic nerves (parasympathetic branches) (Weingartner et al. 1996). One of the main branches of the inferior hypogastric plexus is the cavernous nerve (neurovascular bundle), responsible for erection. A close relationship exists between the cavernous nerve and the lateral surface of the prostate.

3.7  Seminal Vesicles The seminal vesicles are a pair of glands attached to the male genital system, responsible for producing part of the seminal fluid. Each seminal vesicle has an elongated pyriform shape, with a broadened upper end and lower end or neck that joins to the vas deferens (Fig. 3.7). It contains various internal cavities and its wall is mainly formed (about 80%) by smooth muscle tissue (Nguyen et al. 1996). There are two muscle layers, one external and longitudinal and the other internal and circular. Knowledge of the seminal glands is important for surgery and radiology. Anteriorly, each seminal vesical is related to the portion of the bladder corresponding to the trigone vesical. It is possible to analyze the seminal vesicles through digital rectal exam and transrectal ultrasound. Laterally, the seminal vesicles are related Fig. 3.7  Posterior view of the seminal vesicles. 1 ureter, 2 posterior region of the bladder, 3 seminal vesicles, 4 vas deferens, 5 ejaculatory ducts, 6 prostate

1

2 3

4

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with the periprostatic venous plexus. The vascularization of the seminal vesicles is provided by the branches of the internal iliac artery, mainly by the vas deferens artery, inferior vesicle artery, and middle rectal artery. The venous drainage occurs to the periprostatic venous plexus. The lymph of the seminal vesical drains into the internal iliac lymph nodes and the innervation is from the inferior hypogastric plexus.

3.8  Ejaculatory Ducts Each ejaculatory duct is formed by the union at an acute angle of the seminal vesicle and the vas deferens. After a path of 15–20 mm in adults, the ejaculatory ducts open into the prostatic urethra in a small ostium located in the rear part of the seminal colliculus, one to the right and the other to the left of the prostatic utricle. The ejaculatory duct can be divided into three segments: proximal, median, and distal (Nguyen et  al. 1996). The proximal segment is located at the junction with the seminal vesicle. The duct is the right continuation of the seminal vesicle, while the ampoule of the vas deferens enters medially at an acute angle. The smooth muscle fibers of the external longitudinal layer of the seminal vesicle continue along the wall of the ejaculatory duct. The intraprostatic portion or median segment runs on the outside of the prostate surface for 10–15 mm. The distal segment, located in the central region of the prostate, opens into the seminal colliculus (Weingartner et al. 1996).

3.9  Testis and Scrotum The scrotal sack is formed by a skinfold in the perineal region and houses the testes, epididymis, and elements of the spermatic funiculus. The scrotal sack is divided into two independent compartments by a median raphe. Infectious pathologies or buildup of liquids in one of the compartments generally does not disseminate to the other side due to the presence of this anatomical barrier (Parker and Robison 1971). Just beneath the skin is the tunica dartos, which is continuous with the superficial perineal fascia and the superficial fascia of the abdomen. The arrangement of this tunica explains the dissemination of urine from the anterior urethra to the anterior abdominal wall and perineum that occurs in traumas (Netter 1978). Below the tunica dartos is a series of layers that surround the testes. These layers are derived from the abdominal wall, through which each testicle crosses when migrating to the scrotum. The outermost layer is the external spermatic tunica, derived from the external oblique abdominal muscle; the second layer is the cremaster tunica, derived from the internal oblique abdominal muscle; the third layer is the internal spermatic tunica, derived from the transversal abdominal muscle; and the deepest layer is the tunica vaginalis, derived from the peritoneum (Netter 1978).

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After testicular migration, the communication between the testis and tunica vaginalis – the peritoneal-vaginal conduit – closes. If this does not occur, the patient can develop indirect inguinal hernia. The dissection of all the tunicas described above reveals the testicle, which has two extremities, one superior and the other inferior; two lateral edges; and two surfaces, one anterior and the other posterior, which are covered by the epididymis. A testicular appendage is located at the upper end of each testis. The testis is enveloped by a thick tunica with a large quantity of dense connective tissue, the tunica albuginea, which sends septa to the interior of the testis, dividing it into lobes, where the convoluted seminiferous tubules are located (Fig. 3.8). These tubules reach the mediastinal region of the testis, where they become straight and give rise to the testicular network from where the efferent tubules depart, which establish the communication between the testis and head of the epididymis (Parker and Robison 1971).

3.10  Lymphatic Vascularization and Drainage Each testis is irrigated by three arteries: testicular artery, a branch of the right aorta; deferential artery; and cremasteric artery, a branch of the internal iliac artery. These three arteries penetrate the organ in the mediastinal region, where they provide ample communication. Knowledge of the anatomy of the veins that drains into the testes is clinically important due to the pathology of these veins that is one of the main causes of male infertility, varicocele. The testes are drained by the pampiniform venous plexus, which in the region of the deep inguinal ring originates the testicular veins. The left testicular vein opens into the left renal vein and the right testicular vein opens directly into the inferior cava vein (Netter 1978). Fig. 3.8  Visualization of the convoluted seminiferous tubules (1), straight seminiferous tubules (2), and vas deferens (3)

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The testicular veins have several valves distributed throughout their extension. In the region of the fourth lumbar vertebra, each testicular vein divides into two trunks, one lateral and one medial (Fig.  3.9). The lateral trunk is anastomosed with the retroperitoneal veins, mainly the colonic and renal capsular veins, and the medial trunk is anastomosed with the ureteral veins. There is also communication between the two venous trunks (Netter 1978). Varicocele consists of the dilation of the testicular veins, principally on the left side. Its treatment requires surgery and consists of ligature of the varicose veins. One of the factors involved in the relapse after surgery is the anatomic distribution of the testicular veins in the retroperitoneum (Wishahi 1991). The lymphatic drainage of the scrotal sack is performed by the superficial inguinal lymph nodes. The right testis drains into the retroperitoneal lymph nodes, located along the renal pedicle and inferior cava vein, as well as between the inferior cava vein and the aorta. The left testis drains into the lymph nodes located along the left renal hilum and aorta artery. One of the main routes for dissemination of testicular tumors is through the lymphatic system. Knowledge of the lymphatic drainage of the testicles and the scrotal sack is important for proper diagnosis and therapy of these tumors. Fig. 3.9 Abdominal pathway of the gonadal vein. In the region of the fourth lumbar vertebra, the gonadal vein bifurcates into a lateral trunk that anastomoses with the retroperitoneal veins and a medial trunk that drains into the inferior cava vein. VG gonadal vein, m medial trunk, l lateral trunk M

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VG

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3.11  Epididymis The epididymis is the organ responsible for the maturation, storage, and transport of the spermatozoids. It is the first portion of the efferent vein of the testis and is located on the posterolateral face of the testis. It has dilated upper portion, called the head; a central portion, the body; and a lower tapered end, the tail. The head of the epididymis is directly connected to the cranial pole of the testis by the efferent ducts. The tail is linked to the inferior pole of the testis by areolar tissue and a reflection of the tunica vaginalis. A recession of the tunica vaginalis, the sinus of the epididymis or mesorchium, is found between the body of the vas deferens and lateral face of the testis (Netter 1978). There are 12–15 efferent ducts of each testis. They are straight but become sinuous after penetrating the head of the epididymis. In this region, they form wedge-­ shaped masses, the epididymal lobules (Fig.  3.8), and open into the epididymal duct. This duct is tangled and forms the body and tail of the epididymis. The epididymal duct increases in diameter and thickness as it approaches the tail of the epididymis, where it becomes the vas deferens. The epididymal convolutions are maintained by fine areolar tissue and fibrous tissue. The head and body of the epididymis are supplied by the epididymal artery, which originates from the testicular artery, at a variable distance from the epididymis. This artery generally arises from the region of the inguinal canal and forms an anastomotic arch with the deferential artery. The head of the epididymis receives additional supply from the testicular artery. Three arteries contribute to the irrigation of the tail of the epididymis: the epididymal artery, deferential artery, and testicular artery. In some cases there is a fourth artery, a branch of the cremasteric artery, participating in the irrigation of this region (Netter 1978). The veins of the body and tail of the epididymis form an anastomotic layer called the Hoeber epididymal vein. The principal veins of the epididymis join with the pampiniform plexus (Netter 1978). The lymphatic drainage of the epididymis occurs through two pathways: the lymph nodes of the head and body drain to the testicular lymph nodes, while the head drains into the external iliac lymph nodes. The nerves of the testis and epididymis accompany the testicular vessels and are derived from the tenth and eleventh segments of the spinal cord, through the renal and aortic plexuses.

3.12  Vas Deferens The vas deferens is a continuation of the epididymal duct that transports the spermatozoids from the epididymis to the ejaculatory duct. The vas deferens starts in the tail of the epididymis, where it is sinuous, and ascends via the medial side of the epididymis where it is surrounded by the pampiniform plexus, becoming part of the

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spermatic cord. It continues in an upward direction to the external inguinal ring and in this region it is easily palpated. It is the rearmost element with hardest consistency of the spermatic cord. This is the region where the vas deferens is affected during vasectomy. After passing through the inguinal canal, it curves around the inferior epigastric artery and ascends anteriorly to the external iliac artery, returning posteriorly and inferiorly and penetrating the pelvis (Fig. 3.10). It crosses the medial face of the ureter, meets the posterior face of the bladder, and runs in the inferior and medial direction on the medial face of the seminal vesical. In this region, the duct is dilated and sinuous and is called an ampoule, joining the seminal vesicle duct to form the ejaculatory duct. The arterial irrigation of the vas deferens is performed by the deferential artery, a branch of the internal iliac artery. The venous drainage is carried out by a plexus that surrounds the vas deferens.

3.13  Anatomy of the Penis Good knowledge of the penile anatomy is fundamental to comprehend the erection mechanism. The penis is divided into two portions: the root, located in the surface space of the perineum, which is responsible for the attachment and stability of the penis, and the free portion, which composes the largest part of the organ and is

Fig. 3.10  Pathway of the vas deferens; vd vas deferens

bv

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Fig. 3.11 Diagram showing the root and free portion (F) of the penis

L

composed of three erectile structures, the two corpora cavernosa and the corpus spongiosum, which presents a distal expansion covered by skin, the glans penis (Netter 1978) (Fig. 3.11).

3.14  Root of the Penis The root of the penis is formed by a dilated central extremity, the bulb, and two lateral parts, the branches or pillars of the penis (Fig. 3.12). The bulb is located in the interval between the two branches and is attached to the inferior surface of the inferior face of the urogenital diaphragm, from where it continues anteriorly with the corpus spongiosum and is crossed in its central portion by the bulbar urethra and surrounded by the bulbospongiosus muscle (Netter 1978). The contraction of the bulbospongiosus muscle helps the emptying of the urethra in the final phase of urination and during emission of the seminal fluid. The branches of the penis are elongated formations that are adhered to the lower portions of the ischium and pubis, covered by the ischiocavernosus muscles. The contraction of these muscles is responsible for compressing the penile root, leading to an increase in pressure in the corpus cavernosum, which increases the penile rigidity. The contraction of the ischiocavernosus muscles acts to increase penile rigidity mainly during penetration (Claes et al. 1996).

3  Basic Anatomy of the Male Genital System Fig. 3.12 (a) Diagram showing the penile erectile bodies. The urethral bulb can be observed at the root of the penis (b) as well as the penile branches or pillars (p). (b) Section of the penile root showing the bulbar urethra (1); spongiosum body (2) and the cavernosum bodies (3), we can observe the tunica albuginea (4) and the pubic bone (5). (c) Sagittal section of penile body of a fresh cadaver indicating the tunica albuginea (1), the cavernosum body (2), and the penile urethra (3)

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L. A. Favorito and F. J. B. Sampaio

c

3.15  Free Portion of the Penis The free portion of the penis is divided into the body and glans, the latter of which is covered by a skinfold called the foreskin or prepuce. The upper part of the penile body is also called the dorsal region and the lower part, through which the urethra runs, is called the ventral region.

3.16  Glans and Prepuce The glans is the dilated distal portion of the corpus spongiosum (Fig. 3.12). It has a slight elevation that separates it from the body of the penis, called the corona, and is separated from the prepuce by the balanopreputial fold. The prepuce has a vascularized extension from the mucosa that is attached on the ventral portion of the glans, denominated the frenulum of prepuce. The tip of the glans is the location of the external urethral meatus. This region is also the location of the navicular fossa, the second most dilated region of the urethra, only behind the prostatic urethra (Netter 1978). The navicular fossa is better visualized when making a sagittal cut of the glans (Fig. 3.13). The prepuce is a specialized and intensely innervated musculocutaneous tissue that covers and protects the glans. At birth, the glans cannot be exposed because the prepuce’s internal epithelial face is fused with the glans. When reaching 2 or 3 years of age, keratinization cysts break the adherence spots of the prepuce and, together with intermittent erections, enlarge the phimotic ring and allow the exposure of the glans. Some 80–90% of uncircumcised boys can expose the glans after the age of 3 years (Orsola et al. 2000).

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Fig. 3.13  Sagittal section of the penis of a formalized cadaver indicating the navicular fossa (*) of the glandular urethra

Fig. 3.14  The figure shows a 4-year-old patient suffering from phimosis, unable to expose the glans

Physiological phimosis affects 96% of newborns and its incidence diminishes with age. At 3 years of age, 10% of patients still present phimosis, a figure that falls to only 1% at 14 years of age (Shankar and Rickwood 1999) (Fig. 3.14). The natural process prepuce enlargement is subject to alterations due to episodes of balanoposthitis or lesion of the prepuce due to traumatic traction of the ring, leading to the formation of fibrous scar tissue, making exposure of the glans impossible. There are various classifications of the position of the phimotic ring (Elmore et al. 2002; Atilla et al. 1996; Kikiros et al. 1993), but only Kayaba demonstrates the shape and degree of retractability of the prepuce anatomically (Kayaba et al. 1996). According to this classification, there are four groups according to the retraction degree of the prepuce. In group A, the prepuce cannot be retracted at all; in group B, only the urethral meatus can be exposed; in group C, half of the glans can be exposed; and in group D, the exposure of the glans is incomplete due to the presence of adherences in the balanopreputial fold.

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3.17  Body of the Penis The body of the penis is formed by three erectile structures that become engorged with blood during erection, the two corpora cavernosa and the corpus spongiosum. The corpora cavernosa are located on the dorsal region of the penis, i.e., the posterosuperior region, when the penis is erect. The corpus spongiosum is crossed by the urethra and is located in the ventral region of the penis, i.e., the region that is in contact with the perineal region when the penis is flaccid. The corpus spongiosum continues in a dilated distal region to the glans penis. A transversal cut in the body of the penis (Fig. 3.15) enables observing that the erectile bodies are surrounded by a series of structures. Just below the skin is the subcutaneous tissue, called the superficial fascia of the penis. Fig. 3.15 (a) Schematic drawing of a cross section of the body of the penis showing the arrangement of the corpora cavernosa and the tunica albuginea (a). (b) Penis of a fetus in the third gestational trimester cut transversally, indicating the collagen in red. Note the large concentration of collagen fibers in the tunica albuginea (*); Sirius red 40×

a

A

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This superficial fascia contains a small quantity of smooth muscle fibers and is virtually bereft of fat. It continues with the tunica dartos of the scrotal sack and with the fascia of the perineum. Below the fascia of the penis is a continuation of the deep perineal fascia, the deep fascia of the penis, or Buck’s fascia. It is a strong and membranous fascia that surrounds the two corpora cavernosa and the corpus spongiosum (Netter 1978). The deep fascia of the penis is surrounded by dense system of fibers, called the tunica albuginea. The portion of the tunica albuginea that surrounds the corpora cavernosa is thinner than the part that surrounds the corpus spongiosum (Fig. 3.15). Numerous trabeculae originate from the tunica albuginea, dividing the interior of the erectile bodies into cavernous spaces (Goldstein et al. 1985). The tunica albuginea, mainly the portion that surrounds the corpora cavernosa, has an important role in erection. The tunica albuginea of the corpora cavernosa is principally composed of thin collagen bundles containing sparse elastic fibers (Fig. 3.15). Since the tunica albuginea expands during erection, the arrangement of these collagen bundles must allow its expansion. The thickness of the tunica albuginea is around 2–3 mm in the flaccid state and 0.5 mm during erection. The tunica albuginea is almost totally formed by thin collagen fibers. From the deep surface of the tunica, fibrous columns penetrate the corpus cavernosum at different depths. Both the tunica albuginea and the fibrous columns originating from it have undulations and sparse elastic fibers. The tunica albuginea is crossed by veins and nerves. These pass through the interior of the tunica albuginea, which protect the vessels and nerves from compression during erection. Although the tunica albuginea is almost entirely formed by inelastic material, it is able to expand during erection due to the undulations of its collagen fibers in the flaccid state (Goldstein et al. 1985).

3.18  Arteries of the Penis The penis is irrigated by two internal pudendal arteries, branches of the internal iliac (hypogastric) artery. After its various perineal branches, the pudendal arteries combine to form the so-called common penile artery, which divides into three branches: the bulbourethral artery, the dorsal penile artery, and the cavernosal artery. The cavernosal artery is located inside the corpus cavernosum, the bulbourethral artery is responsible for irrigating the corpus spongiosum and urethra, and the dorsal penile artery is located between the tunica albuginea and Buck’s fascia. These penile arteries branch out, giving rise to smaller arteries that run into the cavernous spaces, the helicine arteries (Netter 1978). The cavernous arteries and their helicine branches are surrounded by fibrous sheaths. These sheathes establish connections with the trabeculae of the tunica albuginea. The pressure during erection is very high, so the arteries would be unable to function if they were not surrounded by the fibrous sheaths. This anatomic arrangement probably prevents the arteries from collapsing during erection, when

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the elements of the fibrous web are stretched. The helicine arteries can also establish these connections, when the terminal arterioles open within the cavernous spaces. The smooth muscle bundles can be observed inside the fibrous sheaths of the cavernous arteries, running parallel to the axis of the arteries. In some places, these bundles can be observed perforating the fibrous sheath of the cavernous artery and establishing a connection with the muscle tissue of the parenchyma (Netter 1978). Arterial cushions or pads were described by Conti et al. (1988), and based on these morphological findings a vascular regulation theory was proposed that prevailed for many years. However, in recent years, some authors (Benson et al. 1981; Conti et al. 1988) have cast doubt on these earlier anatomical findings and instead have proposed that those structures are really atherosclerotic alterations of the penile arteries.

3.19  Veins of the Penis The venous drainage of the penile erectile bodies originates in small venules that start in the perisinusoidal spaces located below the tunica albuginea. These venules connect to the emissary veins, which cross the tunica albuginea and open into the circumflex veins, located beside the erectile bodies. The circumflex veins drain into the deep penile dorsal vein, which in turn opens into the periprostatic venous plexus. The circumflex veins have one or more valves. The skin and subcutaneous tissue of the penis are drained by the superficial penile dorsal vein, which generally empties into the greater saphenous vein.

3.20  Lymphatic Drainage of the Penis Knowledge of the lymphatic drainage of the penis is important for comprehension of the dissemination of the epidermoid tumors of this organ. The skin and prepuce drain into the superficial lymph nodes in the inguinal region, located above the fascia lata. The glans and the rest of the penis drain into the deep inguinal lymph nodes, located under the fascia lata, and to the external iliac lymph nodes. The so-called sentinel lymph node of Cabanas is of particular importance in the dissemination of penile tumors. It is located medially across from the great saphenous vein and is generally the first place afflicted in the lymphoid dissemination of penile tumors (Netter 1978).

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3.21  Nerves of the Penis The penis is innervated by the dorsal penile nerves, which are branches of the pudendum nerve, in turn innervating the skin and mainly the glans. The deep branches of the perineal nerves, which enter the bulb and mainly innervate the urethra and cavernous penile nerves, in turn are branches of the inferior hypogastric plexus, responsible for the autonomic innervation of the penis, in particular the penile erectile bodies (Ophoven and Roth 1997). The cavernous nerves are very important in the erection process. They have different anatomical characteristics than other nerves and are located in fibrous tunnels in which numerous fibrous bands establish connections. During erection, when the corpus cavernosum filled with blood is under high pressure, these perineural fibrous tunnels, which are connected to all the fibrous elements of the corpus cavernosum, including the albuginea, stretch and prevent compression of the nerves during erection. The intracavernous pressure during the erect state can be tenfold that of the systolic pressure. In the flaccid penis, the nerves are spiraled or undulated. This, just as occurs with the helicine arteries, permits their elongation and adjustment to the changes in the dimension of the corpus cavernosum during erection (Conti et al. 1988).

References Atilla KM, Dundaroz R, Odabas O, Ozturk H, et al. A nonsurgical approach to the treatment of phimosis: local nonsteroidal anti-inflammatory ointment application. J Urol. 1996;158(1):196–7. Benson GS, McConnell A, Schmidt WA. Penile polsters: functional structures or atherosclerotic changes? J Urol. 1981;125:800–3. Brooks JD. Anatomy of the lower urinary tract and male genitalia. In: Campbell’s urology. 8th ed. New York: Saunders; 2002. p. 41. Brooks JD, Chao WM, Kerr J. Male pelvic anatomy reconstructed from the visible human data set. J Urol. 1998;159:868–72. Claes H, Bijnens B, Baert L.  The hemodynamic influence of the ischiocavernosus muscles on erectile function. J Urol. 1996;156:986–90. Conti G, Virag R, von Niederhausern W. The morphological basis for the polster theory of penile vascular regulation. Acta Anat. 1988;133:209–12. Elmore JM, Baker LA, Snodgrass WT. Topical steroid therapy as an alternative to circumcision for phimosis in boys younger than 3 years. J Urol. 2002;168:1746–7. Goldstein AMB, Meehan JP, Morrow JW, Buckley PA, Rogers FA.  The fibrous skeleton of the corpora cavernosa and its probable function in the mechanism of erection. Br J Urol. 1985;57:574–8. Kayaba H, Tamura H, Kitajima S, Fujiwara Y, Kato T, Kato T. Analysis of shape and retractability of the prepuce in 603 Japanese boys. J Urol. 1996;156:1813–5.

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Kikiros CS, Beasley SW, Woodward AA. The response of phimosis to local steroid application. Pediatr Surg Int. 1993;8:329. Manley CB. The striated muscle of the prostate. J Urol. 1966;95:234–40. McNeal JE, Redwine EA, Freiha FS, Stamey TA.  Zonal distribution of prostatic adenocarcinoma: correlation with histologic pattern and direction of the spread. Am J Surg Path. 1988;12:897–907. Myers RP. Anatomical variation of the superficial preprostatic veins with respect to radical retropubic prostatectomy. J Urol. 1991;145:992–3. Netter F. Reproductive system, vol. 2. New Jersey: CIBA; 1978. Nguyen HT, Etzell J, Turek PJ. Normal human ejaculatory duct anatomy: a study of cadaveric and surgical specimens. J Urol. 1996;155:1639–42. Oerlich TM. The urethral sphincter muscle in the male. Am J Anat. 1980;158:229–46. Ophoven A, Roth S.  The anatomy and embryological origins of the fascia of Denoviliers: a medico-­historical debate. J Urol. 1997;157:3–9. Orsola A, Caffaratti J, Garat JM. Conservative treatment of phimosis in children using a topical steroid. Urology. 2000;56(2):307–10. Parker RM, Robison JR. Anatomy and diagnosis of torsion of the testicle. J Urol. 1971;106:243–7. Sampaio FJB.  Neoplasia prostática: Conceitos anatômicos fundamentais para a compreesão da patologia benigna e maligna. J Bras Urol. 1992;18:121–4. Shankar KR, Rickwood AM. The incidence of phimosis in boys. BJU Int. 1999;84(1):101–2. Weingartner K, Ramaswamy A, Bittinger A, Gerharz EW, et al. Anatomical basis for pelvic lymphadenectomy in prostate cancer: results of an autopsy study and implications for the clinic. J Urol. 1996;156:1969–71. Wishahi MM. Detailed anatomy of the internal spermatic vein and the ovarian vein. Human cadaveric study and operative spermatic venography: clinical aspects. J Urol. 1991;145:780–4.

Chapter 4

Basic Anatomy of the Female Genital Tract Luciano Alves Favorito

4.1  Uterus Anatomy The uterus consists of three layers: the outermost perimetrium, the muscular myometrium, and the innermost endometrium, which is shed during menstruation. The uterus is located in the pelvis, anterior to the rectum and posterior to the bladder (Fig. 4.1), in an anteflexion position in relation to the vaginal axis and flexed in relation to its own axis (Testut 1921; Rouviére 1961). The pelvic viscera in women are covered with peritoneal reflections, which give rise to pouches that have surgical importance. There are three pouches in the female pelvis: (1) pre-vesicle, between the pubis and bladder; (2) vesicouterine, between the bladder and uterus; and (3) rectouterine (Douglas pouch), between the rectum and uterus (Fig.  4.2). The Douglas pouch is particularly important for being the point of greatest declivity of the female peritoneal cavity when the individual is in orthostatic position, leading to accumulation of secretions in this space. The Douglas pouch can be punctured through the vaginal region, which surrounds the lower part of the uterus – the vaginal fornix. Its examination can help in the diagnosis of certain pathologies, such as ectopic pregnancy (Williams et al. 1995). The peritoneum that covers the uterus is called the broad ligament and is an important structure in the lining and attachment of the organ. Beneath the broad ligament there is a tissue that has various thick spots that serve to stabilize the pelvic viscera (Williams et al. 1995). This tissue is called the subperitoneal pelvic connective tissue and gives rise to various ligaments. The two most important for attachment of the uterus are the uterosacral ligament and lateral cervical ligament. The lateral cervical (cardinal) ligament envelops the region just above the uterus cervix and has great surgical importance for being the place where the uterine artery L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_4

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Fig. 4.1  The figure shows a female pelvis with the anatomical relations of the uterus (U) with the rectum (R) and bladder (B)

passes before going to the uterus. This is the point where the uterine artery crosses the ureter and anatomical relationship of great interest when performing hysterectomies (Fig. 4.3) (Netter 1978). Another ligament that is important for uterus attachment, also sectioned during hysterectomy, is the round ligament, a tapered ligament that joins the body of the uterus near the uterine tube to the large lips. This ligament is the main component of the female inguinal canal and is the homologue of the gubernaculum in men. The uterus has four parts: fundus, body, isthmus, and cervix (Fig. 4.4). The fundus is the region located above the uterine tubes; the body composes the major portion of the uterus; the isthmus is a narrow region located between the body and cervix; and the cervix is the lowest region of the uterus, enveloped by the vagina (Testut 1921; Rouviére 1961). The uterine cervix contains the uterine ostium, which establishes the communication between the uterine cavity and vagina. The uterus cervix is the region of greatest clinical concern because it is commonly afflicted by pathologies, such as cervical cancer (Williams et al. 1995).

4.2  Uterine Tubes The uterine (or Fallopian) tubes are bilateral ducts that extend from the uterus to the ovaries, with the function of capturing and conducting the oocyte to the uterine cavity. They permit the spermatozoids to meet the oocyte, so they are the usual place of fertilization and initial division of the ovule (zygote). The tubes are situated in the pelvic cavity, in the upper part and between the two layers of the broad ligament of the uterus. Like the ovaries, they are located approximately 8–10  mm below the upper opening (inlet) of the pelvis. The direction of tubes in each hemipelvis is generally lateral, from the uterus to the uterine end of the ovary, so they pass over

4  Basic Anatomy of the Female Genital Tract Fig. 4.2 (a) Schematic drawing of the relations of the uterus with the pelvic viscera; note the peritoneal reflections in highlight. B bladder, U uterus, and FSD Douglas pouch. (b) Upper view of the pelvis of a formalized female cadaver: U uterus, * Douglas pouch, O ovary, L round ligament

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the mesovaric margin, arching posterolaterally and projecting over the tubal extremity of the uterus and ending on the medial surface and free margin (Kamina 1968). The tubes are covered by the peritoneum, and the latter’s continuation with the pelvic segment occurs through the mesosalpinx, which, by homology with other mesos in the abdomen, rests loosely on them, unlike what occurs in the uterus body, where it is adhered to the organ (Fig. 4.5) (Williams et al. 1995). Four morphologically distinct parts are recognized in each tube: the infundibulum, ampule, isthmus, and uterine (intramural) parts (Fig. 4.5).

52 Fig. 4.3  Crossing of the ureter with the uterine artery. (a) Schematic drawing showing the crossing between the ureter and uterine artery. (b) Cross section of a female cadaver showing the crossing between the uterine artery and ureter

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The uterine tubes are contained in the broad ligament, a peritoneal formation that extends from the pelvis wall to the edge of the uterus edge. They are surrounded by the peritoneum, whose two layers (anterior and posterior) compose the mesosalpinx. The uterine tubes are mobile in relation to the uterus and pelvic walls, following the movements of the uterus during pregnancy and retroversion (Woodruff and Pauerstein 1969). Together with the mesosalpinx, the uterine tubes form the upper edge of the broad ligament, which are located posterior in relation to the surface of the round ligament of the uterus and anterosuperior in relation to the ligament of the ovary itself. Between the uterine tubes and the round ligament, which diverge (the former laterally and the latter laterally), is the ovarian recess (Woodruff and Pauerstein 1969). The mesosalpinx contains the vessels and nerves of the uterine tubes and

4  Basic Anatomy of the Female Genital Tract Fig. 4.4  Uterus anatomy: (a) schematic drawing showing the parts of the uterus: (1) fundus, (2) body, (3) isthmus, and (4) cervix. (b) Frontal cross section of a female pelvis showing the parts of the uterus

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sometimes embryonic vestiges: epoophoron, located laterally, and paraovarian medially (Woodruff and Pauerstein 1969). Beside the mesosalpinx, the uterine tubes are located below the rings of the small intestine, anterolateral to the rectal ampoule and posterolateral to the bladder, maintaining this relation with these organs when they are full. The ampoule and infundibulum are connected to the pelvic wall in front of the ovary and below the iliac vessels (Williams et al. 1995).

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Fig. 4.5  Uterine tube. (a) Diagram of the anatomical relation of the layers of the broad ligament with the uterine tube, ovary, round ligament, vascularization, and ureter. (b) Photo of a female fetus from the second gestational trimester showing the relations of the uterine tube with the pelvic structures

4.3  Ovaries The ovaries are the pair of primary sexual organs of women, which produce oocytes (ovules) and sexual hormones such as estrogen and progesterone. For this reason, they are considered important organs of the feminine reproductive apparatus and are homologues of the testes in men (Williams et al. 1995). The ovaries are located in the pelvic cavity, generally 8–10 mm below the upper pelvic opening, in the so-called ovarian recess. Their major axis has superoinferior, lateromedial, and anteroposterior obliquity. They are found in an anterolateral position in relation to the rectum, posterior to the broad ligament, and 15–20 mm in front of the sacroiliac joint (Bergman et  al. 1988). This location is the result of migration that, like for the testes, starts in the lumbar region medially to the mesonephric duct, but unlike the testes, they are retained in the pelvis definitively, generally in the ninth month of intrauterine life (Williams et al. 1995). The lateral face of the ovary has relations with the external iliac veins, internal iliac veins, and ureters, separated from them only by the lateral pelvic peritoneum, with which it can present important alterations as a result of varied pathologies (Williams et al. 1995). The medial face and free (posterior) edge of the right ovary is connected to the rings of the small intestine, while the left ovary is also connected to the sigmoid. The mesovaric edge, where the blood vessels, lymph nodes, and nerves penetrate and continue accompanying the peritoneum, is one of the attachment points of the ovary, called the ovarian hilum. Each ovary is attached by four ligaments: one on the pelvic wall and three on the ovary itself. These multiple ligaments allow the ovary to move, because they converge to the hilum. This enables each ovary to accompany the ascending and descending movements of the uterus during pregnancy and postpartum. Their traction during surgery permits moving them to a position far from the original one (Williams et al. 1995).

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4.4  Vascularization, Lymph System, and Innervation The main artery responsible for irrigation of the uterus is the uterine artery, a branch of the anterior trunk of the internal iliac artery. The uterine artery passes over the cardinal ligament and ascends through the lateral regions of the uterus to irrigate it (Fig.  4.6). The uterine artery also participates in irrigation of the uterine tubes, ovary, and part of the vagina. The uterine tube vessels originate from the ovarian and uterine trunks, with the following origins: (a) superolaterally from the tubal branch of the ovarian artery (lateral tubal artery) and (b) medially from the tubal branch of the uterine artery. The two arteries (uterine and ovarian) reach the uterine tube at (Koritké et al. 1967; Borell and Fernström 1953). The main arterial irrigation of the ovary is performed by the ovarian artery and right branch of the abdominal aorta artery, arising from the anterolateral contour of the aorta just below the renal arteries, at the level of the L2/L3 intervertebral disk (Machnicki and Grzybiak 1999). The venous drainage is performed by the uterine veins, which accompany the arteries in the direction of the internal iliac vessels (Williams et al. 1995). The uterine lymphatic drainage is of great importance in cancer surgeries. The upper part of the uterine body drains into the lumbar lymph nodes; the lower part of the body drains into the external iliac lymph nodes; the cervix drains into the internal iliac, external iliac, and sacral lymph nodes; and the region near the round ligament drains into the superficial inguinal lymph nodes (Sampson 1937) (Fig. 4.7). The nerve supply, mainly along the ovarian and uterine arteries, follows a similar pattern of distribution. The greater part of each tube has sympathetic and parasympathetic innervation. The vagal nerve fibers reach the lateral half, while the pelvic-­ splanchnic fibers reach the medial half. The sympathetic innervation comes from the tenth thoracic segment and from the second lumbar spinal segment (Chiara 1959; Fig. 4.6  Arterial irrigation of the uterus; note that the uterine artery (AU) is also responsible for irrigation of part of the uterine tube (T) and ovary (O)

T

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Fig. 4.7 Lymphatic drainage of the uterus to lumbar lymph nodes, internal iliac lymph nodes (3); external iliac lymph nodes (4); sacral lymph nodes (5) and inguinal lymph nodes

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Damiani and Capodacqua 1961). The afferent nerve fibers accompany the sympathetic motor innervation, penetrating the spinal marrow through the corresponding dorsal roots. The postganglionic sympathetic fibers and visceral afferent fibers, as well as the preganglionic parasympathetic fibers, are located in the tubal walls.

References Bergman RA, Thompson SA, Afifi AK, Saadeh FA. Compendium of human anatomic variation. Baltimore: Urban & Schwarzenberg; 1988. Borell U, Fernström I. Adnexal branches of uterine artery; arteriographic study in human subjects. Acta Radiol. 1953;40:561–82. Chiara F.  Study of the fine innervation of the female genitália. I.  Uterus. Ann Ostet Ginecol. 1959;81:553–76. Damiani N, Capodacqua A. On the intrinsic innervation of the fallopian tube. Ann Ostet Ginecol. 1961;83:436–46. Kamina P. Anatomie Gynecologique et Obstetricale. Paris: Librairie Maloine; 1968. Koritké JG, Gillet JY, Pietri J. Les artères de la trompe uterine chez la femme. Arch Anat Histol Embryol. 1967;50:47–70. Machnicki A, Grzybiak M.  Variations in ovarian arteries in fetuses and adults. Folia Morphol (Warsz). 1999;58:115–25.

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Netter F. Reproductive system, vol. 2. Summit: CIBA; 1978. Rouviére H. Anatomía humana descriptiva y topográfica. 8th ed. Madrid: Bailly-Bailliere; 1961. Sampson JA.  Lymphatics of mucosa of fimbriae of the fallopian tube. Am J Obstet Gynecol. 1937;33:911–30. Testut L.  In: Livre X, editor. Anatomíe humaine: appareil uro-génital. 7th ed. Paris: Librairie Octave Doin; 1921. Williams PL, Bannister LH, Berry MM, Dyson M, Dussek JE, Ferguson MWJ. Gray’s anatomy. 38th ed. Edinburgh: Churchill-Livingstone; 1995. Woodruff JD, Pauerstein CJ. The fallopian tube. Baltimore: Williams & Wilkins; 1969.

Chapter 5

Testicular Migration Luciano Alves Favorito

5.1  Introduction Testicular descent is a complex process of relevant importance for the comprehension of cryptorchidism (Hutson et  al. 2010). During the human fetal period, the testes migrate from the abdomen to the scrotum traversing the abdominal wall and the inguinal canal between the 15th and the 28th week of postconception (WPC) (Heyns and Hutson 1995; Sampaio and Favorito 1998).

5.2  Phases and Theories of Testicular Migration Testicular migration has two phases: (a) abdominal stage, testicular migration from the abdomen to the internal inguinal ring that begins around the 8th WPC and lasts until the 15th WPC, and (b) inguinal-scrotal stage, transition of the testes through the inguinal canal until their definitive arrival in the scrotum that begins around the 20th WPC and lasts until the 30th WPC (Husmann 2009; Heyns and Hutson 1995; Favorito et al. 2016) (Fig. 5.1). The moment when testicular migration begins is controversial. Backhouse (1982) reports that this process starts at about the 24th week postconception, while Heyns (1987) and Sampaio and Favorito (1998) describe cases where the migration process started as early as the 17th week. An important aspect that various authors report is that the passage of the testis through the inguinal canal occurs very quickly (Heyns 1987; Heyns and Hutson 1995; Sampaio and Favorito 1998). Heyns (1987) found only 2.6% of the testes examined in his sample located in the inguinal canal, L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_5

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Fig. 5.1  Schematic drawing showing the development of the vaginal process and its relationship with the gubernaculum and inguinal canal during the period of growth of the testis. (a) Fetus in the second month of pregnancy, (b) fetus in the third month of pregnancy, (c) last month of gestation, and (d) after birth

while Sampaio and Favorito (1998), in a sample of 71 human fetuses, found 20.5% of the testes located there. Furthermore, 73.3% of these testes were in fetuses with ages between 21 and 25 WPC, indicating that in this period the migration through the inguinal canal intensifies. In the same study, all the fetuses older than 30 weeks already had the testes in the scrotum. Other authors, however, report that testicular migration is only completed after the 32nd week of postconception (Gill and Kogan 1997; Wensing 1988). Asymmetrical testicular migration is a rare process. A theory to explain this occurrence is imbalance in the testicular gubernaculum and the vaginal process (Heyns and Husmann). On the side where the gubernaculum is more developed, the testes migrate faster (Heyns et al. 1986). Studies that refer to asymmetrical testicular migration during the human fetal period are rare. Heyns (1987) observed asymmetry only in 17% of the human fetuses examined, with age ranging from 23 to 31 weeks postconception. In cases of asymmetry in testicular migration during the human fetal period described, he observed that the left testicle was located lower in 70% of the cases. In a recent paper (Favorito and Sampaio 2014) in a study with 164

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Fig. 5.2 Schematic drawing of a fetus aged 23 weeks postconception. We can observe asymmetrical testicular migration. The rigth testis is situated above the internal ring and the right testis is situated in scrotum

human fetuses, the authors concluded that the asymmetrical testicular migration is very rare and when this asymmetrical migration occurs, there is a discrete predominance of migration of the right testis first. A case of asymmetrical testicular migration is observed in the figure below (Fig. 5.2). There are several anatomic and hormonal factors involved in testicular migration. The most accepted ones are (a) a rise in intra-abdominal pressure (Attah and Hutson 1993), (b) the development of the structures near the testis (epididymis, spermatic vases, and deferent ducts) (Hadziselimovic 1984), (c) the stimulus originating in the genitofemoral nerve (Clarnette and Hutson 1996), (d) the hormonal stimulus originating in the placental gonadotropin and the testosterone produced by the fetal testes (Barthold et al. 2000; Husmann and Levy 1995; Nation et al. 2009), and (e) the gubernaculum development (Hutson et al. 2013). We will describe these factors with an emphasis on the study of gubernaculum structure.

5.3  Rise in Intra-abdominal Pressure An old and quite controversial theory of testicular migration is the role of intra-­ abdominal pressure. The contraction of the abdominal wall musculature, the growth of the liver and intestines, and the accumulation of meconium increase the pressure

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inside the fetal abdomen, which according to some authors would favor testicular migration (Attah and Hutson 1993; Frey and Rajfer 1984). Another fact that speaks in favor of this theory is the high incidence of cryptorchidism in patients with abdominal wall defects, such as omphalocele, gastroschisis, and prune belly syndrome (Levard and Laberg 1996; Hassett et al. 2012). This theory, however, does not explain cases of asymmetry in testicular migration, where one testicle migrates normally, while the other is located in the inguinal canal or abdomen (Favorito and Sampaio 2014). An interesting study has shown that intra-abdominal pressure is a supporting factor for testicular migration. The author performed an experiment in which defects were created in the anterior abdominal wall of animals associated or not with the section of the proximal portion of the gubernaculum (Attah and Hutson 1993). What was evident was that there was a significant decrease in testicular migration only in cases where the abdominal wall defect was accompanied by sectioning of the gubernaculum. In cases of isolated defects in the abdominal wall the testicles migrated in 96% of the cases. This experiment demonstrates that abdominal pressure would act only as an auxiliary force in testicular migration, while the gubernaculum and patency of the vaginal process would be of great importance for the orientation of the testicular path during migration.

5.4  Hormonal Stimulus Testicular migration is a complex process mediated by endocrine and mechanical factors. The integrity of the axis between the testis, hypothalamus, and pituitary, which regulates testosterone production, is important for the testicular migration process. Cryptorchidism is a common event in pathologies on this axis, such as hypogonadotropic hypogonadism and 5-alpha reductase deficiency (Husmann and Levy 1995; Fu et al. 2004). Testosterone appears to play an active role in testicular migration, inducing the development of important structures for testicular migration such as the vaginal process, the vas deferens, the epididymis, the inguinal canal, and the scrotum. Another mechanism of action of testosterone would be through stimulation of the genitofemoral nerve, which would induce the production of calcitonin gene-related peptide (CGRP) that acts by stimulating the development of the testicular gubernaculum. Fetal and placental gonadotropins are also implicated in the process of testicular migration. These substances act by stimulating the production of testicular androgens, which induce the growth and development of the vas deferens, the epididymis, the vaginal process, and the gubernaculum itself (Nation et  al. 2009). It is well known that treatment of cryptorchidism with gonadotropins induces testicular migration at levels ranging from 25% to 55% of cases (Gapany et al. 2008). Another endocrine substance involved in testicular migration is descendin (Hutson et  al. 2010). This androgen-independent secreted substance in the testis would play an important role in the growth of the gubernaculum mesenchymal cells. The

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gubernaculum would therefore be one of the fetal structures implicated in testicular migration, most modified by hormonal action (Elder et al. 1982).

5.5  Stimulus Originating in the Genitofemoral Nerve According to this theory, fetal androgens masculinize the spinal nucleus of the genitofemoral nerve and then the nerve itself (Su et  al. 2012). This masculinization results in an increase in the number of motoneurons in this region with consequent increase in secretion of the calcitonin gene-related peptide (CGRP). The importance of this mechanism is corroborated by experimental models where the sectioning of the genitofemoral nerve leads to cryptorchidism (Heyns and Hutson 1995). Increased CGRP levels lead to a rhythmic contraction of the testicular gubernaculum that would induce its migration to the scrotum (Clarnette and Hutson 1996). The site of action of the CGRP is the neuromuscular junction. In experimental animals such as rodents, there is an abundance of musculature, fortifying this hypothesis (Clarnette and Hutson 1996), but the human gubernaculum is basically composed of an abundant extracellular matrix with high concentration of glycosaminoglycans (Heyns 1987; Costa et  al. 2002), so this theory of CGRP-induced traction in humans is debatable.

5.6  D  evelopment of the Structures Near the Testis (Epididymis, Spermatic Veins, and Deferent Ducts) Growth and development of the hormone-mediated vas deferens, testicular vessels, epididymis, and vaginal process are necessary for the testis to migrate to the scrotum. The vaginal process, as seen in the Attah experiment (Attah and Hutson 1993), acts as a guide for the testicle to reach the scrotum. Alterations in the vaginal process may be associated with cryptorchidism (Husmann and Levy 1995). An interesting and controversial theory proposed by Hadziselimovic (1984, 2017) suggests that the epididymis would be responsible for testicular migration through its peristaltic and secretory activity in the second gestational trimester. There would be changes in the gravitational center of the epididymis, causing the testis to migrate along with it. This theory would explain some cases of cryptorchidism, where the epididymis is separated and situated lower than the testis (Elder et al. 1982). Some observations support the theory described above. The epididymis precedes the testis in the scrotum; the epididymis is in a privileged position to influence testicular migration; for it is anatomically connected to the gubernaculum, which is in turn attached to the testis and scrotum. Morphological and functional changes occur in the epididymis at the time of migration in certain animal species (Scorer and Farrington 1971; Hutson and Hasthorpe 2009).

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5.7  Gubernaculum Testis The gubernaculum seems to be the most important anatomical structure in the testicular migration process, by means of contraction and shortening, thus imposing traction strength on the testis facilitating the transition of the testis through the inguinal canal (Heyns and Hutson 1995; Favorito et al. 2014). In the abdominal phase of testicular migration, the gubernaculum enlarges to hold the testes near the groin, regulated by insulin-like 3 (INLS-3) (Husmann 2009). INSL-3 is secreted by the Leydig cells and controls gubernaculum swelling via its receptor, LGR8 (leucine-rich repeat containing G protein-coupled receptor 8, also known as GREAT or relaxin receptor 2), a process resulting in thickening of the gubernaculum because of increases in water, glycosaminoglycan, and hyaluronic acid content (Husmann 2009; Heyns and Hutson 1995; Favorito et al. 2014). In the second phase of testicular migration (transition of the testes through the inguinal canal), the gubernaculum migrates across the pubic region to reach the scrotum. The androgens stimulate growth and differentiation of the muscular part of the gubernaculum bulb, which facilitates the movement of the gubernaculum through the inguinal region by the traction resulting from this growth (Allnutt et al. 2011; Hutson and Hasthorpe 2009). The gubernaculum has its own nerve supply, the genitofemoral nerve (GFN), descending on the anteromedial surface of the psoas muscle from L1-L2 segments (Heyns and Hutson 1995; Favorito et al. 2014). The second phase of testicular descent is regulated by androgens and calcitonin gene-related peptide (CGRP), released from the sensory nucleus of the genitofemoral nerve (GFN) (Clarnette and Hutson 1996; Fu et al. 2004). In rodents, the active proliferation of the gubernacular tip and cremaster muscle, the muscle’s rhythmic contraction, and the chemotactic gradient provided by the CGRP together result in migration of the testes into the scrotum. The importance of this mechanism is corroborated by experimental models where the sectioning of the genitofemoral nerve leads to cryptorchidism (Husmann and Levy 1995; Hutson and Hasthorpe 2009). The gubernaculum starts to develop in the human fetus during the sixth week of gestation, the same period when the germinative cells are arriving at the genital ridge (Wensing 1988; Heyns and Hutson 1995). In the eighth week of gestation, the testis and mesonephros are linked to the posterior abdomen wall by a peritoneal fold. As the mesonephros degenerates, the portion of this fold cranial to the testis, called the diaphragmatic ligament, also degenerates, turning into the cranial portion of the gonadal mesentery. This structure is called the caudal gonadal ligament, which gives rise to the gubernaculum testis (Heyns 1987; Wensing 1988). Cranially, the gubernaculum approaches the mesonephric duct, while distally it approaches the inguinal region. At this moment, the future inguinal canal is still only a space in the musculature of the anterior abdominal wall, where only mesenchyme tissue exists. In this region, the genital branch of the genitofemoral nerve crosses the abdominal wall and descends to the scrotum where it will innervate the

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cremaster muscle and, subsequently, in the caudal to cranial direction, will provide the nerve supply to the gubernaculum (Wensing 1988; Heyns and Hutson 1995). At about the eighth week of gestation, a portion of the epithelium starts a small invagination from the coelomic cavity, across from the gubernaculum, slowly penetrating its mesenchymal substance. This invagination occurs bilaterally and is considered the start of the vaginal process. Some authors consider this phenomenon to be “active,” involving the invasion of the gubernaculum by mesothelial cells (Wensing 1988), while others advocate that this phenomenon is “passive” and secondary to the increase in intra-abdominal pressure (Frey and Rajfer 1984). The growth of the vaginal process divides the gubernaculum into three parts: (a) the main gubernaculum, which corresponds to the portion covered by the visceral layer of the peritoneum of the vaginal process; (b) the vaginal gubernaculum, which corresponds to the portion that externally surrounds the parietal portion of the vaginal process; and (c) the infra-vaginal gubernaculum, corresponding to the caudal region of the gubernaculum, which has not been invaded by the vaginal process (Wensing 1988) (Fig. 5.3). Both the gubernaculum and vaginal process change in harmony during testicular migration. The maintenance of this undifferentiated mesenchyme along the inguinal canal and scrotum is essential for the downward extension of the vaginal process to Fig. 5.3 Schematic drawing based on Wensing’s 1988 work showing the gubernaculum and the testis. On the left side of the scheme, the gubernaculum was sectioned longitudinally and its divisions are pointed after the formation of the vaginal process. 1, testis; 2, gubernaculum; 2′, gubernaculum itself; 2″, infra-vaginal gubernaculum; 2″′, vaginal gubernaculum; 3, vaginal process; and 4, testicular artery (study done in pigs)

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occur, during which it follows the pathway created by dilation of the gubernaculum, forming the canal through which the testis will reach the scrotum (Wensing 1988; Heyns and Hutson 1995). The gubernaculum is a cylindrical structure, covered by a peritoneum on all sides except the posterior, where the testicular vessels and vas deferens pass (Fig. 5.4). Macroscopically, it looks like the Wharton’s jelly of the umbilical cord. Histologically, it is composed of undifferentiated cells with elongated shape, surrounded by a large quantity of extracellular material, where it is impossible to identify smooth or striated muscle cells except in its distal end and in the peripheral portion (Costa et al. 2002) (Fig. 5.5).

Fig. 5.4  Testicular migration during the human fetal period: (a) image of a male fetus aged 20 weeks postconception with the testis situated in abdomen; (b) fetus aged 21 weeks postconception with the testis situated in abdomen, in this figure the inguinal canal was opened and we can observe the gubernaculum (G) implantation in the inguinal canal; (c) schematic drawing showing the migration of the testis by the inguinal canal, IR internal ring, ER external ring and (d) a fetus aged 28 weeks postconception with the testis situated in scrotum. LT left testis, RT right testis, and E epididymis

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Fig. 5.5  Macroscopic aspect of gubernaculum testis: (a) in a fetus aged 20 weeks postconception the testis (T) was separate together with the epididymis (E) and the gubernaculum (G). We can observe that the gubernaculum looks like the Wharton’s jelly of the umbilical cord (*) Testicular vessels. (b) In the same fetuses, a histological preparation was done, and we can observe that the gubernaculum is composed of undifferentiated cells with elongated shape, surrounded by a large quantity of extracellular material, HE ×40

5.7.1  Proximal Gubernaculum The proximal portion of the gubernaculum is adhered to the lower pole of the testis and to the epididymis (Fig. 5.6). During testicular migration, these structures move through the inguinal canal as a single unit (Beasley and Hutson 1988). According to previous studies (Johansen and Bloom 1988), in this situation the proximal gubernaculum is always adhered to the end of the vaginal process. Jackson et al. (1987), studying 60 boys submitted to orchiopexy, found the gubernaculum adhered to the

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Fig. 5.6 Schematic drawing showing relationships between the testis, the epididymis, and the proximal portion of the gubernaculum. T testis, E epididymis, and G gubernaculum

lower testicular pole in all cases, but did not mention its relationship with the epididymis. Other studies have shown that changes in the proximal insertion of the gubernaculum are associated with epididymal anomalies and can contribute to the occurrence or cryptorchidism (Favorito et al. 2000). Attah and Hutson (1993), in a study with rats, demonstrated the importance of the integrity of the proximal portion of the gubernaculum for proper testicular migration. The proximal portion is important by uniting the scrotal region and serves as a guide for testicular migration. In this

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experiment, the authors performed transection of the proximal gubernaculum. After this procedure, the testicular migration was only completed in 26 of the 70 rats (37%), and of these animals, 24 showed testicular torsion. The results of this study indicate that the proximal gubernaculum is important both to guide testicular migration and to limit the mobility of the testes and prevent testicular torsion. Abe et al. (1996), in a study of 44 patients with cryptorchidism, found an elongated epididymis in 42.5% of the cases. Of the patients with elongated epididymis, alterations in the proximal gubernaculum were found in 73.9% of the cases. In other study of human fetuses, the authors found a low rate of epididymal anomalies (2.75%) (Favorito and Sampaio 1998).

5.7.2  Distal Gubernaculum The insertion site of the gubernaculum during testicular migration is variable. Studies have shown that in the period before the end of testicular migration, the distal gubernaculum is not firmly attached to the scrotum (Heyns 1987; Heyns and Hutson 1995; Favorito et al. 2014). While the testis is in the abdomen, the gubernaculum is firmly attached to the inguinal canal (Heyns and Hutson 1995) (Fig. 5.7). The insertion site of the distal gubernaculum is one of the factors involved in testicular ectopia (Nightingale et al. 2008). Several papers have reported that the distal gubernaculum has six extensions: abdominal, pubo-penile, femoral, perineal, contralateral scrotal, and scrotal (Heyns and Hutson 1995; Ramareddy et al. 2013). It is speculated that these branches of the distal gubernaculum exist during the start of fetal development and disappear during testicular migration (Heyns and Hutson 1995; Ramareddy et al. 2013). If any of these extensions of the distal portion persist, the individual can develop testicular ectopia (Ramareddy et al. 2013). Various theories have been proposed to explain testicular ectopia. The most accepted are as follows: (a) failure of the gubernaculum to dilate the inguinal canal, enabling the testis to migrate through other pathways and not reach the scrotum (Nightingale et al. 2008); (b) invasion of the gubernaculum by abdominal wall fascia near the inguinal canal, blocking the passage of the testis to the scrotum and diverting it to ectopic sites (Heyns 1987; Ramareddy et al. 2013); and (c) the existence of multiple distal insertions of the gubernaculum testis, guiding the testis to the main ectopic sites (Heyns and Hutson 1995; Nightingale et al. 2008). The most accepted theory to explain testicular ectopia is the existence of multiple distal insertions of the gubernaculum. According to this theory, proposed by Lockwood in the nineteenth century (Nightingale et  al. 2008), the gubernaculum presents six distal insertion sites, in decreasing order of frequency: scrotal, interstitial (abdominal), femoral, perineal, transverse (contralateral scrotal), and pubo-penile. Multiple distal insertions of the gubernaculum exist at the beginning of fetal development and disappear during testicular migration (Ramareddy et  al. 2013; Nightingale et al. 2008). Pubo-penile testicular ectopia is considered the rarest form of this anomaly, but in one study with fetuses, the only two cases of anomalous insertion of the gubernaculum were located in the pubo-penile region (Favorito et al. 2003).

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Fig. 5.7  Gubernaculum testis during testicular migration: (a) a fetus aged 20 weeks postconception: we can observe the testes in abdominal position and the gubernaculum situated inside the inguinal ring. (b) A fetus aged 22 weeks postconception: testes are situated in inguinal canal and we can observe the gubernaculum testis; (c) a male fetus aged 21 weeks postconception. The inguinal canal was dissected, revealing the enlargement of the distal portion of the gubernaculum. RT right testis, LT left testis, G gubernaculum, P penis, and S scrotum

5.7.3  S  tructure of the Gubernaculum During Testicular Migration The different parts of the gubernaculum undergo varied changes during testicular migration. The vaginal and infra-vaginal portions become proportionally longer as the testis starts to descend to the scrotum. At the same time, their diameter increases, a fact considered by Heyns (1987) to be one of the most important mechanisms for dilating the inguinal canal to allow the testis to pass.

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The gubernaculum’s growth is divided into two phases, triggered by different hormonal stimuli (Nation et al. 2009; Wensing 1988). In the first phase, its volume increases, and in the second phase, it decreases in size, coinciding with the complete descent of the testis (Van Vlinsingen et al. 1989). The cremaster muscle presents structural alterations during this period as well (Lie and Hutson 2011). This muscle allows rhythmic contraction to guide the testis into the scrotum in rats and in humans, leading to eversion of the distal portion of the gubernaculum and contributing to its migration to the scrotum (Lie and Hutson 2011) The first phase is marked by pronounced cell multiplication and accumulation of glycosaminoglycans, mainly hyaluronic acid. These substances act as hydrophilic agents and raise the quantity of water. There is also an increase in the amount of extracellular material, explaining the low cell density found at some points (Heyns et al. 1990). The presence of myoblasts intensifies and there are changes in the number and arrangement of the collagen fibers and alterations of the elastic system. In the second phase, the gubernaculum shrinks, particularly its length, normally accompanied by descent of the testis. This phenomenon appears to be androgen dependent and brings substantial degradation of the glycosaminoglycans previously accumulated in the extracellular material, with consequent dehydration of this space and condensation of the gubernaculum (Costa et al. 2002). Although no estimates are available of the degree of shortening, some authors believe this acts together with other factors, causing the gubernaculum to convey the testis to the scrotum (Wensing 1988; Heyns and Hutson 1995). Understanding the relationship between regression of the gubernaculum and descent of the testis is vital to comprehension of how androgens control testicular migration. Studies have demonstrated an association between androgen deficiency, on the one hand, and failed regression of the gubernaculum and cryptorchidism on the other. In this situation, the gubernaculum appears to act as an obstacle to testicular descent (Elder et al. 1982; Barthold et al. 2000). Differences between the proximal and distal portions of the gubernaculum have been reported. In one study, in fetuses aged 15–15 WPS the authors observed a greater number of muscle cells in the distal portion, arranged in isolated groups, while in the proximal portion the muscle tissue was present in smaller quantity and was arranged peripherally. With increasing age, the quantity of muscle tissue was found to decrease. In fetuses between 28 and 29 WPC, the authors observed a large quantity of elastic fibers and almost no muscle fibers in the entire gubernaculum (Costa et al. 2002) (Fig. 5.8). In the early fetal period (15 and 16 WPC), when the testes are still in the abdomen, the connective tissue is loose and poor in collagen. As the gestational time increases and the testes migrate from the abdominal cavity, the connective tissue becomes progressively denser and richer in collagen (Fig.  5.9). In fetuses with 28–29 WPC, the gubernaculum presents very dense organization of the collagen fibers and predominance of fibroblasts, with sharp directional orientation of the fibers and cells (20). Likewise, the reticular fibers, which are arranged more loosely in the gubernaculum at the beginning of the fetal period (15 and 16 WPC), are very dense in the gubernaculum of fetuses with 28 and 29 WPC (Costa et al. 2002).

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Fig. 5.8  Gubernaculum testis: (a) the figure shows the testis and the gubernaculum of a male fetus aged 21 weeks postconception with both testes situated in the abdomen. (b) Photomicrograph of a male fetus aged 15 weeks postconception, with both testes situated in the abdomen. A low concentration of collagen and elastic fibers in the gubernaculum can be observed. HE, 400×. (c) Photomicrograph of a male fetus aged 35 weeks postconception, with both testes situated in the scrotum. A condensation of the gubernaculum with a large amount of collagen and elastic fibers can be observed. Masson’s trichrome, 200×

Changes in the tissue components of the gubernaculum during the fetal period have been reported in various experimental studies (Heyns et al. 1990). The relative presence of muscle tissue appears to be one of the factors that affect the traction the gubernaculum exerts on the testis during its migration (Heyns 1987; Heyns and Hutson 1995). At the start of the fetal period, a good deal of muscle tissue is present, but it starts to diminish with time, while the elastic tissue, which is sparse at the beginning, is markedly higher when the fetus reaches 25 WPC. At 28 and 29 WPC, under normal circumstances the testes have already completed their migration and are located in the scrotum. At this point in gestational age, a previous study (Costa

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Fig. 5.9  Collagen system distribution in gubernaculum. Gubernaculum sample of a male fetus aged 22 weeks postconception and the testis in abdominal position examined under scan electron microscopy (SEM). In this preparation, the collagen fibers are visible. In this group the collagen ranged in thickness from 0.2 to 0.4 μm. The fibers are densely packed in parallel undulating arrays. Original ×5000; the scale bar represents 1 μm Fig. 5.10 Photomicrograph of a 3-year-old boy with both testes situated in inguinal canal. A condensation of the gubernaculum with a large amount of collagen and elastic fibers can be observed. Masson’s trichrome, 200×

et al. 2002) observed very sparse muscle fibers and a large quantity of elastic fibers in the gubernaculum, especially in the distal portion. The connective tissue of the gubernaculum undergoes remodeling, so that at the end of migration it has essentially become a fibrous structure, rich in collagen and elastic tissue (Costa et al. 2002). The tissue changes in the gubernaculum testis during the fetal period suggest that it plays an active role in testicular migration (Fig. 5.10).

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In summary, the morphological alterations of the extracellular matrix of the gubernaculum likely lead to a reduction of its length and volume. Although there is not sufficient evidence to estimate the degree of shortening, this change probably acts synergistically with other factors, causing the gubernaculum to guide the testis to the scrotum (Costa et  al. 2002). Testicular descent is therefore a complex and multifactor event, and cryptorchidism should be viewed as a pathology with multiple etiologies. One of the factors involved in cryptorchidism is the failure of the gubernaculum to migrate all the way to the scrotum (Hutson et al. 2010). Structural studies conducted in patients with cryptorchidism reveal significant changes in the gubernaculum’s structure, with a higher quantity of fibrous tissue and lower concentration of collagen than in the fetal gubernaculum (Soito et al. 2011). The influence of fetal androgens on the fetal gubernaculum’s development is very important for the alterations of this structure, and the changes in its secretions can be one of the factors involved in cryptorchidism (Nation et al. 2009). For example, in a study analyzing the structure of the gubernaculum in patients treated with hCG, the authors observed that the gubernacular components change significantly when submitted to hormonal treatment, with an increase in the concentration of elastic and striated muscle fibers and a decrease in the volumetric density of collagen (El Zoghbi et al. 2007).

5.8  Conclusions 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 muscles and nerves in the human gubernaculum is very poor and the gubernaculum presents significant structural modifications during testicular migration in human fetuses. The gubernaculum of patients with cryptorchidism has more fibrous tissue and less collagen, and when patients are submitted to hormonal treatment, the gubernacular components change significantly.

References Abe T, Aoyama K, Gotoh T, Akiyama T, Iwamura Y, Kumori K. Cranial attachment of the gubernaculum associated with undescended testes. J Pediatr Surg. 1996;31:652–5. Allnutt B, Buraundi S, Farmer P, Southwell BR, Hutson JM, Balic A. The common fetal development of the mammary fat pad and gubernaculum. J Pediatr Surg. 2011;46:378–83. Attah AA, Hutson JM.  The role of intra-abdominal pressure in cryptorchidism. J Urol. 1993;150:994–6.

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Backhouse KM.  Embryology of testicular descent and maldescent. Urol Clin North Am. 1982;9:315–25. Barthold JS, Kumasi-Rivers K, Upadhyay J, Shekarriz B, Imperato-Mcginley J. Testicular position in the androgen insensitivity syndrome: implications for the role of androgens in testicular descent. J Urol. 2000;164:497–501. Beasley SW, Hutson JM.  The role of the gubernaculum in testicular descent. J Urol. 1988;140:1191–3. Clarnette TD, Hutson JM.  The genitofemoral nerve may link testicular inguinoescrotal descent with congenital inguinal hernia. Aust N Z J Surg. 1996;66:612–7. Costa WS, Sampaio FJB, Favorito LA, Cardoso LEM. Testicular migration: remodeling of connective tissue and muscle cells in human gubernaculum testis. J Urol. 2002;167:2171–6. El Zoghbi CS, Favorito LA, Costa WS, Sampaio FJB. Structural analysis of gubernaculum testis in cryptrochid patients submitted to treatment with human chorionic gonadotrophin. Int Braz J Urol. 2007;33:223–9. Elder JS, Isaacs JT, Walsh PC. Androgenic sensitivity of the gubernaculum testis: evidence for hormonal/mechanical interactions in testicular descent. J Urol. 1982;127:170–6. Favorito LA, Sampaio FJ. Anatomical relationships between testis and epididymis during the fetal period in humans (10 to 36 weeks postconception). Eur Urol. 1998;33:121–3. Favorito LA, Sampaio FJ. Testicular migration chronology: do the right and the left testes migrate at the same time? Analysis of 164 human fetuses. BJU Int. 2014;113:650–5. Favorito LA, Sampaio FJB, Javaroni V, Cardoso LEM, Costa WS. Proximal insertions of gubernaculum testis in normal human fetuses and in boys with cryptorchidism. J Urol. 2000;164:792–4. Favorito LA, Klodja CAB, Costa SW, Sampaio FJB. Distal insertions of gubernaculum testis and their relationships with testicular ectopia. J Urol. 2003;170:554–3. Favorito LA, Costa SF, Julio JRHR, Sampaio FJ. The importance o-f the gubernaculum in testicular migration during the human fetal period. Int Braz J Urol. 2014;40:722–9. Favorito LA, Bernardo FO, Costa SF, Sampaio FJ. Is there a trans-abdominal testicular descent during the second gestational trimester? Study in human fetuses between 13 and 23 weeks post conception. Int Braz J Urol. 2016;42:558–63. Frey HL, Rajfer J. Role of the gubernaculum and intra-abdominal pressure in the process of testicular descent. J Urol. 1984;131:574–9. Fu P, Layfield S, Ferraro T, Tomiyanma G, Hutson JM, Otvos L Jr. Synthesis, conformations, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3. J Pept Res. 2004;63:91–8. Gapany C, Frey P, Cachat F, Gudinchet F, Jichlinski P, Meyrat BJ, Ramseyer P, Theintz G, Burnand B. Management of cryptorchidism in children guidelines. Swiss Med Wkly. 2008;138:492–8. Gill B, Kogan S. Cryptorchidism. Current concepts. Pediatr Clin North Am. 1997;44:1211–27. Hadziselimovic F. Mechanism of testicular descent. Urol Res. 1984;12:155–7. Hadziselimovic F. On the descent of the epididymo-testicular unit, cryptorchidism, and prevention of infertility. Basic Clin Androl. 2017;27:21. Hassett S, Smith GH, Holland AJ. Prune belly syndrome. Pediatr Surg Int. 2012;28:219. Heyns CF. The gubernaculum during testicular descent in the human fetus. J Anat. 1987;153:93–112. Heyns CF, Hutson JM. Historical review of theories on testicular descent. J Urol. 1995;153:754–67. Heyns CF, Husmann HJ, De Klerk DP. Hyperplasia and hypertrophy of the gubernaculums during testicular descent in the fetus. J Urol. 1986;135(5):1043–7. Heyns CF, Husmann HJ, Werely CJ, De Klerk DP. The glycosaminoglycans of the gubernaculum during testicular descent in the fetus. J Urol. 1990;143:612–7. Husmann DA.  Testicular descent: a hypothesis and review of current controversies. Pediatr Endocrinol Rev. 2009;6:491–5. Husmann DA, Levy JB. Current concepts in the pathophysiology of testicular undescent. Urology. 1995;46:267–76. Hutson JM, Hasthorpe S. Abnormalities of testicular descent. Cell Tissue Res. 2009;322:155–8. Hutson JM, Ballic A, Nation T, Southwell B. Cryptorchidism. Semin Pediatr Surg. 2010;19:215–24.

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Hutson JM, Southwell BR, Li R, Lie G, Ismail K, Harisis G, Chen N. The regulation of testicular descent and the effects of cryptorchidism. Endocr Rev. 2013;34:725–52. Jackson MB, Gough MH, Dudley NE.  Anatomical findings at orchiopexy. Br J Urol. 1987;59:568–71. Johansen TEB, Bloom GP. Histological studies of gubernaculum testis taken during orchiopexies. Scand J Urol Nephrol. 1988;22:107–8. Levard G, Laberg JM. The fate of undescended testes in patients with gastroschisis. Eur J Pediatr Surg. 1996;7:163–5. Lie G, Hutson JM. The role of cremaster muscle in testicular descent in humans and animal models. Pediatr Surg Int. 2011;27:1255–65. Nation TR, Balic A, Southweel BR, Newgreen DF, Hutson JM. The hormonal control of testicular descent. Pediatr Endocrinol Rev. 2009;7:22–31. Nightingale SS, Al Shareef YR, Hutson JM.  Mythical ‘tails of lockwood’. ANZ J Surg. 2008;78:999–1005. Ramareddy RS, Alladi A, Siddappa OS.  Ectopic testis in children; experience with 7 cases. J Pediatr Surg. 2013;48:538–41. Sampaio FJB, Favorito LA. Analysis of testicular migration during the fetal period in humans. J Urol. 1998;159:540–2. Scorer CG, Farrington GH.  Congenital deformities of the testis and epididymis. London: Butterworths e Co; 1971. Soito ICS, Favorito LA, Costa WS, Sampaio FJB, Cardoso LEM. Extracellular matrix remodeling in the human gubernaculum during fetal testicular descent and in cryptorchidic children. World J Urol. 2011;29:535–40. Su S, Farmer PJ, Li R, Sourial M, Buranundi S, Bodemer D, Southwell BR, Hutson JM. Regression of the mammary branch of the genitofemoral nerve may be necessary for testicular descent in rats. J Urol. 2012;188:1443–8. Van Vlinsingen JMF, Koch CAM, Delpech B, Wensing CJG. Growth and differentiation of the gubernaculum testis during testicular descent in the pig: changes in the extracellular matrix, DNA content, and hyaluronidase, β-glucuronidase, and β-N-acetylglucosaminidase activities. J Urol. 1989;142:837–45. Wensing CJG. The embriology of testicular descent. Horm Res. 1988;30:144–52.

Chapter 6

Basic Research Applied to Undescended Testis Luciano Alves Favorito

6.1  Introduction Cryptorchidism is one of the most common congenital anomalies among males, with a rate between 2% and 5% of full-term births, a rate that can reach 30% in premature babies (Gill and Kogan 1997; Lee and Houk 2013; Hutson and Thorup 2015). This condition exposes the testis to a temperature about 2° centigrade higher than the temperature of the scrotum, which can usually cause damage to the germinal epithelium and may lead to future infertility to the patient, as well as being associated with a higher incidence of testicular cancer (Gill and Kogan 1997; Kollin et al. 2006; Mikuz 2015).

6.2  Etiology Several factors are involved in cryptorchidism: genetic, hormonal, and anatomical (Virtanen et al. 2007). The risk of cryptorchidism is 3.5 times higher in patients with siblings with the disease and 2.5 times higher if the father has had cryptorchidism (Hutson et al. 2010). Undescended testis is a very common finding in sexual differentiation disorders in patients with low birth weight and prematurity (Virtanen et  al. 2007). Several environmental factors also appear to be associated with the undescended testis and other male genital anomalies with hypospadias. Some of these factors are exposure to organophosphates, maternal smoking, and maternal diabetes (Mathers et al. 2009). L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_6

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Testicular migration is a complex process mediated by endocrine and mechanical factors. The integrity of the axis between the testis, hypothalamus, and pituitary, which regulates testosterone production, is important for this process. Cryptorchidism is a common event in pathologies on this axis, such as hypogonadotropic hypogonadism and 5-alpha reductase deficiency (Hutson et al. 2010). Testosterone appears to play an active role in testicular migration, inducing the development of important structures for testicular migration such as the vaginal process, the vas deferens, the epididymis, the inguinal canal, and the scrotum. Another mechanism of action of testosterone would be through stimulation of the genitofemoral nerve, which would induce the production of calcitonin gene-related peptide (CGRP) that acts by stimulating the development of the testicular gubernaculum. Fetal and placental gonadotropins are also implicated in the process of testicular migration. These substances act by stimulating the production of testicular androgens, which induce the growth and development of the vas deferens, the epididymis, the vaginal process, and the gubernaculum itself (Heyns and Hutson 1995). It is well known that the treatment of cryptorchidism with gonadotropins induces testicular migration at levels ranging from 25% to 55% of cases (Cortes et al. 2003). Another endocrine substance involved in testicular migration would be descendin. This androgen-independent secreted substance in the testis would play an important role in the growth of the gubernaculum mesenchymal cells. The gubernaculum would therefore be one of the fetal structures implicated in testicular migration, most modified by hormonal action. Problems with the hypothalamus-pituitary axis are associated with cryptorchidism, which explains the high incidence of this anomaly in patients with Prader-­ Willi syndrome, Kallmann syndrome, pituitary hypoplasia, and anencephaly.

6.3  Cryptorchidism Classification Cryptorchidism can be unilateral or bilateral and is usually more frequent on the right side (Ashley et al. 2010). In general 80% of the testicles are palpable and 20% not palpable, being in the abdominal cavity or absent (Kaefer 2004). Undescended testis may be classified according to its position along the normal course of testicular migration or at ectopic sites. The testis may be located in the abdomen, inguinal canal, external inguinal ring (prepubic), or retractable (Heyns and Hutson 1995).

6.4  Rectractile Testis Retractile testis is defined as a supra-scrotal testis that can be manipulated into the scrotum and will remain there without traction until the cremasteric reflex acts (La Scala and Ein 2004) (Fig. 6.1). The management of this condition is still controversial, including observation to adolescence or early surgical correction. Recent articles have recommended only observation of retractile testis cases (Tekgül et al. 2012; Keys and Heloury 2012). In more than 70% of patients with

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Fig. 6.1  Retractile testis: A 7-year-old patient with left retractile testis. We can observe the retractile testis at rest (a) and after groin stimulation (b)

retractile testis, the condition evolves favorably without the need for surgery (La Scala and Ein 2004). However, a 25% risk exists of these testes ascending and becoming cryptorchidic in adolescence (Agarwal et al. 2006). Structural and ultrasound studies have demonstrated morphological alterations in the germinal epithelium and Sertoli cells of retractile testes (Han et al. 1999; Cinti et  al. 1993). A study of young adults who had retractile testis during prepuberty showed that only 28.8% of the patients had normal sperm count (Caucci et al. 1997). Retractile testis has generally been considered a normal variant, because usually the affected testicle completes its migration to the scrotum during adolescence (Lee et  al. 1999). Patients with retractile testis should be periodically examined until adolescence or the end of testicular migration to the scrotum (Ito et al. 1986). The treatment of retractile testis remains controversial. The more common indication for surgery in retractile testis was given if there is any decrease in testicular volume and when retractile testes ascended. In the present paper we studied only truly retractile testis.

6.4.1  Cremaster Muscle in Retractile Testis The cremaster muscle forms one of the layers of the testicle and derives from the internal oblique muscle of the abdomen (Bingol-Kologlu et  al. 2001) (Fig.  6.2). Previous papers had shown that the cremaster muscle appears to differentiate from the gubernacular tip during elongation to the scrotum (Baker et  al. 2001). The

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Fig. 6.2 Cremaster muscle. An 8-year-old patient submitted to surgical procedure to correct retractile testis. We can observe the cremaster muscle during the surgery

cremaster muscle retracts the testicle in response to genitofemoral stimulus (L1– L2). This cremasteric reflex is absent at birth but generally becomes active at around 3 months of age and should be present in all neurologically normal children after 2 years of age (Bingol-Kologlu et al. 2001). The period between infancy and the start of puberty is when the testes show greatest retractability, due to the low hormone indices in this phase (Kolon et al. 2004). The cremasteric reflex can cause the testicle to retract to the inguinal canal region, where it can remain for an extended period (Bingol-Kologlu et al. 2001). Hyperactivity of the cremaster muscle appears to be the main factor involved in the occurrence of retractile testis, but recent studies have cast doubt on the assumption that structural alterations in the cremaster muscle are responsible for the origin of inguinoscrotal pathologies such as cryptorchidism and inguinal hernia (Keys and Heloury 2012). These studies have demonstrated important differences in the cremaster muscle in patients with cryptorchidism, indicating that in these cases a primary muscular alteration occurs associated with defects in the innervation of the cremaster muscle (Han et al. 1999). Analysis of the nerves of the cremaster muscle of patients with retractile testis also revealed significant differences. These patients had a lower concentration of

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nerve fibers compared to those with inguinal hernia, but no differences in relation to the group with cryptorchidism. The innervation of the cremaster muscle and the gubernaculum testis originates in the genitofemoral nerve (Heyns and Hutson 1995). This nerve, through androgenic regulation and the influence of the calcitonin gene-related peptide (CGRP), plays an important role during the testicular migration process, by neuromuscular junction stimulation of the rhythmic contractions of the cremaster muscle during the fetal period.

6.4.2  E  pididymal Anomalies and Vaginal Process in Rectractile Testis In recent papers it was confirmed that retractile testis is not a normal variant and instead an inguinoscrotal anomaly (Anderson et al. 2017). The cremaster muscle in retractile testis presented a lesser concentration of muscle and nerve fibers compared to the patients with inguinal hernia, but similar morphology to that of patients with cryptorchidism (Fig. 6.3). In a previous study in which surgery was performed on 28 retractile testes, the authors found processus vaginalis patency in 21.4% of the cases (Anderson et al. 2017). These findings confirm that the chance of patients with retractile testis present patent processus vaginalis is not negligible. In the control group composed of fetuses in which the testes had completed their migration, patency was only observed in 4% of the cases, a much lower rate than in the patients with retractile testis (Anderson et al. 2017). Cryptorchidism can be associated with various anatomical anomalies, but ­epididymal anomalies and patency of the processus vaginalis are among the most frequent (Fig.  6.4). Epididymal anomalies are associated with cryptorchidism in over one-third of these cases (Barthold and Redman 1996; Mollaeian et al. 1994). Fig. 6.3  Retractile testis histology. Cremaster muscle of a 3-year-old patient with cryptorchidism. Masson ×100

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Fig. 6.4  Anomaly in retractile testis. A 4-year-old patient with supra-scrotal testicle showing a type V anatomic relation between the testicle and epididymis – total disjunction of epididymis and testicle. Presence of a testicular appendix (AP) and patency of the vaginal process (PV). T testicle, E epididymis

Another study showed that individuals without cryptorchidism have a very low incidence of epididymal anomalies (Favorito and Sampaio 1998). Furthermore, human fetuses without apparent anomalies present epididymal anomalies in less than 3% of the cases, regardless of the testicular position (Kolon et  al. 2004). Epididymal anomalies can be classified as disjunction or atresia (Favorito and Sampaio 1998) and can be associated with infertility. Patients with disjunction anomalies (head, tail, or total disjunction) can present a longer distance between the testis and epididymis, the region called the mesorchium (Kolon et al. 2004; Favorito and Sampaio 1998). Testicular torsion can be intravaginal or extravaginal. Intravaginal testicular torsion can occur because of an anomaly in the implantation of the tunica vaginalis (bell clapper deformity) or due to the presence of an elongated mesorchium because of disjunction anomalies of the epididymis (Favorito et al. 2004). Therefore, patients suffering from epididymal anomalies face a higher risk of developing intravaginal testicular torsion (Favorito et al. 2004). The rate of epididymal anomalies in patients with retractile testis is not well defined in the literature. Anderson et al. (2017) observed that 14% of the patients with retractile testis submitted to orchiopexy presented epididymal anomalies. In three cases we observed tail disjunction, an anomaly where the mesorchium is elongated, and in one case there was total disjunction between the testis and epididymis, a situation associated with infertility and also increased size of the mesorchium.

6.5  Ectopic Testis The insertion site of the distal portion of the gubernaculum would be one of the factors involved in testicular ectopia (Heyns and Hutson 1995). Previous studies reported that the distal portion of the gubernaculum would have six extensions:

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abdominal, pubic-penile, femoral, perineal, contralateral, and scrotal (Heyns and Hutson 1995). It is speculated that these branches of the distal portion of the gubernaculum would exist during early fetal development and disappear during the testicular migration process (Heyns and Hutson 1995). If any of these distal portion extensions persist, the individual could develop an ectopic testis (Cromie 1978).

6.6  Rates of Cancer and Infertility The types of testicular tumor most commonly associated with cryptorchidism are seminoma and embryonic carcinoma with an incidence 35–40 times higher than in the general population (Gapany et  al. 2008). About 10% of testicular tumors are associated with cryptorchidism, and the risk of tumors increases in the highest testis (Gapany et  al. 2008) (Fig.  6.5). The risk of tumor occurrence increases in non-­ operated patients and in patients operated at older ages and usually occurs between 20 and 40 years of age (Docimo et al. 2000; Walsh et al. 2007). Infertility has an increased incidence on cryptorchidism due to germinal epithelial and Sertoli cell changes. The risk of infertility is six times higher in patients with bilateral cryptorchidism than in patients with unilateral cryptorchidism (Chung and Brock 2011). Unilateral cryptorchidism is associated with azoospermia in about 13% of cases, whereas in bilateral cryptorchidism this rate can reach over 80% in untreated bilateral cases (Kaefer 2004). Several histological changes may occur in the contralateral topic testis in unilateral cryptorchidism cases (Kaefer 2004).

6.7  Associated Anomalies in Undescended Testis Cryptorchidism can be associated with various anatomical anomalies, but epididymal anomalies and patency of the vaginal process are among the most frequent (Scorer and Farrington 1971; Barthold and Redman 1996; Lee and Houk 2013). Epididymal anomalies are associated with cryptorchidism with a very variable incidence in the literature: from 36% to 79% (Mollaeian et  al. 1994; Favorito et  al. 2006) (Fig. 6.6). The occurrence of inguinal hernias associated with cryptorchidism is due to the persistence of the vaginal process (Johansen 1987; Lao et al. 2012). The vaginal process (PV) is a conduit that extends from the peritoneum to the scrotum and is covered by a coelomic epithelium. This conduit is usually obliterated after the end of the testicular migration (Johansen 1987; Lao et al. 2012). In cases where the vaginal process does not close, the child may develop inguinal hernia or communicating hydrocele. Knowledge of anomalies associated with cryptorchidism is relevant in clinical practice, both to prevent accidents during orchidopexy and to counsel and predict infertility in the future, such as in cases of epididymis atresia and total disjunction between the testis and the epididymis (Han and Kang 2002; Rachmani et al. 2012).

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Fig. 6.5  The figure shows examples of testicle tumors associated with cryptorchidism. (a) Computed tomography showing abdominal mass in a 23-year-old patient with abdominal cryptorchidism. (b) During laparotomy it was observed testicular mass diagnosed as seminoma. (c) A 25-year-old patient with testicle at the inguinal canal referring pain and increase of local volume. (d) Surgical exploration showed tumoral mass in cryptorchid testicle. Pathology revealed a seminoma

EAs are frequently found in cryptorchidism (Scorer and Farrington 1971; Kim et al. 2015; Sharma and Sen 2013). The abdominal testicles present a higher index of these anomalies (Lee and Houk 2013; Han and Kang 2002). Previous studies on fetuses and children without cryptorchidism have demonstrated an incidence of EAs below 4% (Turek et al. 1994; Favorito and Sampaio 1998). Turek et al. (1994) question the high incidence of EAs associated with cryptorchidism (Scorer and Farrington 1971; Mollaeian et al. 1994; Kubota et al. 2014). The author considers this a consequence of the lack of definition of the normal pattern of the anatomy of the epididymis in the several studies. In a paper (Favorito et al. 2018) with the same standard proposed by Turek et al. (1994) to analyze the

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b E T

Fig. 6.6  Epididymal anomalies in undescended testis. (a) Schematic drawing of epididymal anomalies of disjunction in undescended testis. E epididymis, T testis. (b) A 4-year-old patient with the left testis in inguinal canal. We can observe a total disjunction between the testis and epididymis

relationship between the testis and the epididymis found an incidence of more than 30% of this type of anomaly (disjunction and/or atresia) in patients with cryptorchidism, confirming the high incidence of EAs in cryptorchidism. The timing of vaginal closure is still unknown. Studies suggest that at birth there would be patency of PV in up to 80% of boys, with progressively lower rates during the first year of life (Burgmeier et al. 2014). In a significant number of adult men, the PV is never obliterated; however, in the majority of these cases, there is no development of indirect inguinal hernia (Scorer and Farrington 1971; Johansen 1987). In a study of 137 patients with cryptorchidism, it was evidenced that there was no significant difference in the patency of PV in relation to the age of the patients (Favorito et al. 2005). The patency of PV in patients with cryptorchidism ranges from 21.3% to 81.3% (Aggarwal et al. 2012). In our study, PV was found in more than 66% of cases of cryptorchidism and 23% of fetuses, a significant difference. Regarding testicular position, we observed that all abdominal testicles had patent PV and that the testicles located in the canal presented a PV patency index of 64%, which was higher than the PV patency found in the supra-scrotal testis and testis fetal diseases. Patients with cryptorchidism with patent PV have a higher EA index than in cases where the PV is closed (Scorer and Farrington 1971; Mollaeian et al. 1994; Kim et al. 2015). The index of EAs in patients with cryptorchidism and patent PV varies from 50% to 80% (Scorer and Farrington 1971; Favorito et al. 2006). Of the 72 testicles with patent PV in our study, 27 (37.5%) had EAs and 12 (33.33%) had EAs in the 36 testicles that had the occluded PV, a difference that was not significant. This fact discordant to several studies in the literature can be justified by virtue of the type of classification used to determine epididymal anomalies; in our studies

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we used the classification that is currently accepted in the literature (Sampaio and Favorito 1998; Favorito et al. 2006; Turek et al. 1994). However, when analyzing and comparing cases of EAs in fetuses and patients (72.5% of PV patency) with cases of normal epididymis anatomy in both groups (53.12% of PV patency), the difference was significant, which suggests and confirms the association between EAs and PV patency. Testicular and epididymal appendices have been considered congenital anomalies (Johansen 1987); however, some studies report that these structures are present in most normal individuals (Rolnick et al. 1968). The functions of testicular appendages are controversial: they can control the amount of serous fluid in the vaginal tunica space. A classification was proposed to analyze the testicular appendices: (I) absence of testicular and epididymal appendages, (II) presence of a testicular appendix, (III) presence of appendix of the epididymis, and (IV) presence of testicular appendix and epididymis (Favorito et al. 2004) (Fig. 6.7). Testicular appendices (TAs) presents a significantly lower incidence in patients with cryptorchidism, which could indicate a possible role of TAs in the process of testicular migration (Josza et  al. 2008). In a previous study, it was observed that 62% of testicles with cryptorchidism had testicular appendages (Favorito et  al. 2018), a much larger number than the 24% reported by Jozsa et al. (2008). Josza et al. (2008) in an elegant histologic study analyzed 37 appendix testes that were collected intraoperatively. The great majority of the population analyzed by Jozsa was Caucasian. In this paper we analyzed more than 100 testes of patients with a great variety of ethnicities. The great difference between our paper and Jozsa could be explained by geographical or racial cause. Are there some kind of differences in testicular appendix incidence in the different ethnicities? It is possible, but we need more prospective papers to confirm this hypothesis. We did not find a significant difference in the number of appendages in the testicles with cryptorchidism in relation to the control group, nor did we find a significant difference in the incidence of appendices in relation to testicular position in patients with cryptorchidism. Tostes et al. (2013) also did not observe difference in the number of TAs in the testicles of patients with cryptorchidism in relation to control group. This study showed that the TAs in undescended testes had a larger quantity of elastic fibers and smaller quantity of smooth muscle cells and predominance of type III collagen (Fig. 6.8). The collagen matrix at the testicular appendices in patients with cryptorchidism is disrupted or degraded, rather than fibrotic, which is consistent with higher hydrostatic pressure. This finding suggests that undescended testis has histologic alteration in TAs (Tostes et al. 2013). In the testes where the TAs were present, 73% had the patent PV and 28% EAs. When comparing the incidence of EAs and the patency of PV in appendage patients with patients without appendices, we observed that the presence of appendices is associated more frequently with the patency of PV and EAs. The novelty of this report is the involvement of the investigation of testicular appendages among the previously studied parameters, such as the position of the cryptorchid testis, the presence of the abnormalities of the epididymis, and patency of the vaginal process.

6  Basic Research Applied to Undescended Testis Fig. 6.7 Testicular appendices. (a) Schematic drawing showing the localization of testicular appendices; T testis, E epididymis, VD vas deferens; 1 testicular appendix, 2 epididymal appendices, 3 paradidymis, and 4 superior and inferior vas aberrans of Haller. (Figure of the paper of Favorito et al. 2004, reprinted with permission). (b) We can observe in a 2-year-old boy during the orchidopexy a testicular appendix; * testicular appendix; T testicle, E epididymis

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88 Fig. 6.8 Testicular appendix structure. (a) Histology of testicular appendix of a 4-year-old patient with undescended testis. Masson ×40. (Figure of Tostes et al. 2013, reprinted with permission). (b) Histology of epididymal appendices of a 3-year-old patient with hydrocele. Masson ×40

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Several anomalies are associated with cryptorchidism, but epididymal anomalies and inguinal hernias are among the most common (Scorer and Farrington 1971; Gill and Kogan 1997; Sharma and Sen 2013). Epididymal anomalies are associated with cryptorchidism very variable in the literature: from 36% to 79% of cases (Mollaeian et al. 1994; Barthold and Redman 1996; Favorito et al. 2006). Knowledge of the most common epididymal anomalies can prevent accidents during orchidopexy surgery, such as injury to an elongated epididymis (Koff and Scaletscky 1990). The occurrence of inguinal hernias associated with cryptorchidism is due to the persistence of the vaginal process (Shrock 1971; Johansen 1987; Lao et al. 2012). The vaginal process is a blind-bottomed pouch that extends from the peritoneum to the scrotum and is lined with celiac epithelium (Backhouse 1982). This conduit usually becomes obliterated after the completion of testicular migration (Shrock 1971; Johansen 1987; Lao et al. 2012). In cases where the vaginal process does not close, the child may develop inguinal hernia or communicating hydrocele.

6.8  Diagnosis Clinical examination plays a fundamental role in the diagnosis of cryptorchidism. On inspection, the inguinal region and contralateral testis should be evaluated. Examination of the scrotum usually shows the absence of the testis (Fig.  6.9). During palpation, the inguinal region should be assessed by an increase in abdominal pressure with the patient lying down and sitting. Palpation of the contralateral testis is of importance. Imaging should be ordered only when the clinical examination has any doubts regarding the location and presence of the testis. Ultrasonography (USG) has diagnostic utility mainly in the testes located in the inguinal canal in obese patients, being of little accuracy in the diagnosis of non-palpable testes (Tasian and Copp 2011). Computed tomography and an expensive exam that emits ionizing radiation have a high false-negative index in the investigation of the abdominal testes, thus being restricted to the investigation of cryptorchidism. Nuclear magnetic resonance (MRI) is an expensive examination and has a lower false-negative index than CT for abdominal testis investigation; however it has low sensitivity in identifying the non-palpable testis, but has good diagnostic rates in hydrocele and testicular torsion (Krishnaswami et al. 2013) (Fig. 6.10). Laparoscopy is the exam of choice for the diagnosis of non-palpable testis. It is a diagnostic and therapeutic method. Diagnostic laparoscopy has greater sensitivity and specificity than any other method in diagnosing impalpable testis. Laparoscopy has an advantage over open surgery as it better identifies the anatomy, viability, and location of the impalpable testis (Aggarwal et al. 2012).

90 Fig. 6.9  Clinical exam of a patient with cryptorchidism showing an “empty” left hemiscrotum. This patient had inguinal cryptorchidism

Fig. 6.10  A 5-year-old patient with left testicular hydrocele. The MRI shows the liquid in vaginal tunica

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6.9  Ideal Age for Cryptorchidism Treatment Changes in fertility are considered the main consequence of cryptorchidism. Current studies show that if treatment is performed between 6 months and 1 year of age, these changes can be minimized. A study using hormonal treatment for cryptorchidism demonstrated that treatment before 1 year of age led to higher rates of germinal epithelium preservation (Schwentner et al. 2006). In an elegant clinical study where patients were divided into two groups, one treated at 9 months of age and one at 3 years, it was shown that surgery performed at 9 months had greater benefits on cryptorchidism testis growth (Kollin et al. 2006)

6.10  Clinical Treatment The use of clinical treatment still has much controversy in the literature, but is still used in several centers. This type of treatment should not be administered before 1 year of age due to side effects (Cortes et al. 2003). Hormone treatment stimulates testosterone production. LHRH analogs stimulate LH production and are administered as a nasal spray at a dose of 1.2 mg/day for 4 weeks with a success rate of 30–60% (Cortes et al. 2003). Human chorionic gonadotropin (Pregnyl® or Profasi HP® – 1000, 1500, 2000 UI) stimulates testosterone production and is administered at a daily dose of 72 hours in the following schedule: up to 2 years, 4500 ui; 2–5 years, 6000 ui; 6–10 years, 8000 ui; and above 10 years, ineffective. The success rate of this drug is around 15–50% of cases, with higher indices in testicles in the outer ring or retractable (Cortes et al. 2003). The main side effects of this treatment are premature closure of the epiphyses, development of secondary sexual characteristics, aggressive behavior, and swelling and enlargement of the scrotum and penis.

6.11  Impalpable Testis About 20% of cryptorchidism testes are impalpable and about 20% of non-palpable testes are absent. The exam is fundamental for the diagnosis, but the hormonal stimulus test is of great importance. In the unilateral impalpable testis, hormonal stimulus testing with hCG or LHRH analog is performed; if the testis remains impalpable, an inguinal US should be performed; and if the USG does not demonstrate the testis, diagnostic laparoscopy is indicated (Ashley et al. 2010). If the non-palpable testis is bilateral, testosterone and USG of the inguinal region are measured. After the testosterone dosage is done, the hormone stimulation test with hCG is performed; if after the test the testosterone increases, this is a sign that

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the testis is present and laparoscopic is indicated; however, if the test is negative laparoscopic should be performed anyway to confirm testicular agenesis (Aycan et al. 2006; Ashley et al. 2010).

6.12  Surgical Treatment The purpose of surgical treatment is to place the testis in the scrotum without atrophy or recurrence. The surgery should be performed between 6 and 12 months of age and has success rates around 90% (Cortes et  al. 2003; Docimo et  al. 2000). Surgical complications are more common in cases of testicular anomalies, small or short vessel testicles (Henna et al. 2005). In case the testicles are not palpable, laparoscopic or open surgical exploration is necessary, allowing to identify the morphology and anatomical locations of the testis, vas deferens, and testicular vessels and thus select the most appropriate surgical technique for treatment. Therefore, therapeutic approach is essentially surgical and should preferably be performed until 18 months of life or as soon as the diagnosis is made (Kolon et al. 2014). Surgical therapy is performed through orchidopexy or orchiectomy. In the first the testes are repositioned and fixed in the scrotum after the creation of a dartos pouch, while in the second they are surgically removed. Both surgical treatments can be performed either laparoscopically or openly (Elyas et al. 2010). Orchiectomy is adopted in cases of unilateral cryptorchidism in which the affected testis is atrophic, there is atresia or small length of the vas deferens, and the testicular veins are located in retroperitoneum because they are too short or in the presence of intra-abdominal testis in postoperative pubertal patients, associated with a contralateral testis without anatomical and morphological changes (Kolon et al. 2014). However, in post-pubertal children, there is controversy regarding the adoption of this approach since it would damage the patient’s quality of life and has a higher risk of postoperative mortality, and there is a consensus to do so in cases of increased risk of testicular neoplasia (Chung and Lee 2015). In patients with intra-abdominal testicles one of the follow three surgical techniques can be applied: conventional orchidopexy, laparoscopic orchidopexy, onestage Fowler-Stephens orchidopexy, and two-stage Fowler-Stephens orchidopexy. In the three types of techniques, surgery consists of locating the testis, dissecting it near the spermatic cord, acquiring free tension, and then repositioning it near the scrotum. When performed open, a medial inguinal incision is made to the anterior superior iliac spine to the external oblique fascia to allow exploration of the peritoneal cavity. Advantages of performing these techniques by laparoscopy include improved visualization, extensive vascular dissection ability to vessel origin, lower morbidity, and ability to create a medial inner ring to the lower epigastric vessels and perform a straight course to the scrotum (Elyas et al. 2010). Conventional orchidopexy is performed when the testis has a low location and the testicular vessels and

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Fig. 6.11  Orchidopexy steps. (a) Transverse incision of inguinal region, (b) staged access identifying the testicle and epididymis located at the inguinal canal, (c) blunt dissection of scrotal wall creating a space for testicle positioning (“neo-pouch”), (d) fixation of testicle at the dartos tunic

vas deferens are long enough to be repositioned next to them in the scrotum (Fig. 6.11).

6.13  Laparoscopic Orchidopexy Surgical treatment depends on the location of the testis. Most orchidopexies can be performed through inguinal and even scrotal access. Laparoscopy is often used to diagnose the non-palpable testis, assessing testicular position if the testis is atrophic or absent (Aggarwal and Kogan 2014; Kurz 2016). Laparoscopy allows simultaneous performance of orchidopexy with high success rates (Castilho-Ortiz et al. 2014).

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Fig. 6.12  Testicular fetal vascularization. Male fetus aged 22 weeks postconception; we can observe the left testis in abdominal position and the testicular and deferential arteries

6.14  Fowler-Stephens Surgery Fowler-Stephens orchidopexy, both one- and two-stage procedures, is adopted when the testis is located high and the testicular vessels are too short to be fixed in the scrotum. The vessels are ligated and the blood supply to the testis is preserved via collateral circulation and proceeding with the repositioning and fixation of the testis (Cherian et al. 2014). In one-stage Fowler-Stephens technique, the ligature and fixation of the testis are performed in a single surgical time. In the two-stage procedure, the first stage is the surgical treatment performed to ligate the testicular vessels and the second stage is the repositioning and fixing of the testis to the scrotal pouch after 3–6 months of the first stage (Aggarwal and Kogan 2014; Kurz 2016). Whenever possible, primary orchidopexy is adopted, which has the highest efficiency rate. Comparison of the two Fowler-Stephens techniques shows that the two-stage technique has higher success rates (Cherian et al. 2014; Kolon et al. 2014). Previous fetal studies have shown that the incidence of extra-numerical arteries in fetuses with testicles located in the abdomen is rare (Wafer) and in cases of surgery by the Fowler-Stephens technique the testicular vascularization would be maintained by deferential and cremasteric arteries (Fig. 6.12).

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Aggarwal H, Kogan BA, Feustel PJ. One third of patients with a unilateral palpable undescended testis have a contralateral patent processus. Pediatr Surg. 2012;47:1711–5. Anderson KM, Costa SF, Sampaio FJ, Favorito LA. Do retractile testes have anatomical anomalies? Int Braz J Urol. 2017;42:803–9. Ashley RA, Barthold JS, Kolon TF. Cryptorchidism: pathogenesis, diagnosis, treatment and prognosis. Urol Clin North Am. 2010;37:183–93. Aycan Z, Ustunsalih-Inan Y, Cetinkaya E, Vidinlisan S, Ornek A.  Evaluation of low-dose hCG treatment for cryptorchidism. Turk J Pediatr. 2006;48:228–31. Backhouse KM.  Embryology of testicular descent and maldescent. Urol Clin North Am. 1982;9:315–25. Baker LA, Silver RI, Docimo SG. Cryptorchidism. In: Gearhart JP, Rink RC, Mouriquand PDE, editors. Pediatric urology. Philadelphia: Saunders; 2001. p. 738–53. Barthold JS, Redman JR. Association of epididymal anomalies with patent processus vaginalis in hernia, hydrocele and cryptorchidism. J Urol. 1996;156:2054–6. Bingol-Kologlu M, Tanyel FC, Anlar B, et al. Cremasteric reflex and retraction of a testis. J Pediatr Surg. 2001;36:863–7. Burgmeier C, Dreyhaupt J, Schier F. Comparison of inguinal hernia and asymptomatic patent processus vaginalis in term and preterm infants. J Pediatr Surg. 2014;49:1416–8. Castillo-Ortiz J, Muñiz-Colon L, Escudero K, Perez-Brayfield M.  Laparoscopy in the surgical management of the non-palpable testis. Front Pediatr. 2014;8(2):28. Caucci M, Barbatelli G, Cinti S. The retractile testis can be a cause of adult infertility. Fertil Steril. 1997;68:1051–8. Cherian A, Desai D, Kiely E, Pierro A, Drake D, De Coppi P, Cross K, Curry J, Smeulders N.  Testicular outcome following laparoscopic second stage Fowler-Stephens orchidopexy. J Pediatr Urol. 2014;10:186–92. Chung E, Brock GB. Cryptorchidism and its impact on male fertility: a state of art review of current literature. Can Urol Assoc J. 2011;5:210–4. Chung JM, Lee SD. Individualized treatment guidelines for postpubertal cryptorchidism. World J Mens Health. 2015;33:161–6. Cinti S, Barbatelli G, Pierleoni C, Caucci M.  The normal, cryptorchid and retractile prepurberal human testis: a comparative morphometric ultrastructural study of 101 cases. Scanning Microsc. 1993;7:351–8. Cortes D, Thorup J, Lindenberg S, Visfeldt J.  Infertility despite surgery for cryptorchidism in childhood can be classified by patients with normal or elevated follicle-stimulating hormone and identified at orchidopexy. BJU Int. 2003;91:670–4. Cromie WJ.  Congenital anomalies of the testis, vas epididymis, and inguinal canal. Urol Clin North Am. 1978;5:237–52. Docimo SG, Silver RI, Cromie W. The undescended testicle: diagnosis and management. Am Fam Physician. 2000;62:2037–44. Elyas R, Guerra LA, Pike J, DeCarli C, Betolli M, Bass J, Chou S, Sweeney B, Rubin S, Barrowman N, Moher D, Leonard M. Is staging beneficial for Fowler-Stephens orchiopexy? A systematic review. J Urol. 2010;183:2012–8. Favorito LA, Sampaio FJB.  Anatomical relationships between testis and epididymis during the fetal period in humans (10 to 36 weeks postconception). Eur Urol. 1998;33:121–3. Favorito LA, Cavalcante AG, Babinski MA. Study on the incidence of testicular and epididymal appendages in patients with cryptorchidism. Int Braz J Urol. 2004;30:49–52. Favorito LA, Costa WS, Sampaio FJ. Relationship between the persistence of the processus vaginalis and age in patients with cryptorchidism. Int Braz J Urol. 2005;31:57–61. Favorito LA, Costa WS, Sampaio FJ. Analysis of anomalies of the epididymis and processus vaginalis in human fetuses and in patients with cryptorchidism treated and untreated with human chorionic gonadotrophin. BJU Int. 2006;98:854–7. Favorito LA, Anderson KM, Costa SF, Costa WS, Sampaio FJ. Structural study of the cremaster muscle in patients with retractile testis. J Pediatr Surg. 2018;53:780–3.

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Gapany C, Frey P, Cachat F, Gudinchet F, Jichlinski P, Meyrat BJ, Ramseyer P, Theintz G, Burnand B. Management of cryptorchidism in children guidelines. Swiss Med Wkly. 2008;138:492–8. Gill B, Kogan S. Cryptorchidism. Current concepts. Pediatr Clin North Am. 1997;44:1211–27. Han CH, Kang SH. Epididymal anomalies associated with patent processus vaginalis in hydrocele and cryptorchidism. J Korean Med Sci. 2002;17:660–2. Han SW, Lee T, Kim JH, Choi SK, Cho NH, Han JY. Pathological difference between retractile and cryptorchid testes. J Urol. 1999;162:878–80. Henna MR, Del Nero RG, Sampaio CZ, Atallah AN, Schettini ST, Castro AA, Soares BG. Hormonal cryptorchidism. J Urol. 2005 Mar;173(3):974–7. Heyns CF, Hutson JM. Historical review of theories on testicular descent. J Urol. 1995;153:754–67. Hutson JM, Thorup J. Evaluation and management of the infant with cryptorchidism. Curr Opin Pediatr. 2015;27:520–4. Hutson JM, Balic A, Nation T, Southwell B. Cryptorchidism. Semin Pediatr Surg. 2010;19:215–24. Ito H, Kataumi Z, Yanagi S, Kawamura K, Sumiya H, Fuse H, et al. Changes in the volume and histology of retractile testes in prepubertal boys. Int J Androl. 1986;9:161–9. Johansen TEB.  Anatomy of the testis and epididymis in cryptorchidism. Andrologia. 1987;19:565–9. Josza T, Csizy I, Kutasy B, Cserni T, Flasko T. Decreased incidence of appendix testis in cryptorchidism with intraoperative survey. Urol Int. 2008;80:317–20. Kaefer M. Diagnosis and treatment of the undescended testicle. In: Pescovitz O, Eugster E, editors. Pediatric endocrinology. Philadelphia: Lipincottt Williams &Wilkins; 2004. p. 255–74. Keys C, Heloury Y. Retractile testes: a review of the current literature. J Pediatr Urol. 2012;8:2–6. Kim SO, Na SW, Yu HS, Kwon D.  Epididymal anomalies in boys with undescended testis or hydrocele: significance of testicular location. BMC Urol. 2015;24(15):108. Koff WJ, Scaletscky R.  Malformations of the epididymis in undescended testis. J Urol. 1990;143:340–3. Kollin C, Hesser U, Ritzén EM, Karpe B. Testicular growth from birth to two years of age, and the effect of orchidopexy at age nine months: a randomized, controlled study. Acta Paediatr. 2006;95:318–24. Kolon TF, Patel RP, Huff DS. Cryptorchidism: diagnosis, treatment, and long-term prognosis. Urol Clin North Am. 2004;31:469–80. Kolon TF, Herndon CD, Baker LA, Baskin LS, Baxter CG, Cheng EY, Diaz M, Lee PA, Seashore CJ, Tasian GE, Barthold JS.  Evaluation and treatment of cryptorchidism: AUA guideline. J Urol. 2014;192(2):337–45. Krishnaswami S, Fonnesbeck C, Penson D, McPheeters ML.  Magnetic resonance imaging for locating nonpalpable undescended testicles: a meta-analysis. Pediatrics. 2013;131:e1908–16. Kubota M, Nakaya K, Arai Y, Ohyama T, Yokota N, Nagai Y. The area and attachment abnormalities of the gubernaculum in patients with undescended testes in comparison with those with retractile testes. Pediatr Surg Int. 2014;30:1149–54. Kurz D. Current management of undescended testes. Curr Treat Options Pediatr. 2016;2:43–51. La Scala GC, Ein SH.  Retractile testis: an outcome analysis on 150 patients. J Pediatr Surg. 2004;39:1014–7. Lao OB, Fitzgibbons RJ Jr, Cusick RA. Pediatric inguinal hernias, hydroceles, and undescended testicles. Surg Clin North Am. 2012;92:487–504. Lee PA, Houk CP. Cryptorchidism. Curr Opin Endocrinol Diabetes Obes. 2013;20:210–6. Lee T, Han SW, Lee MJ, Kim JH, Choi SK, Cho NH, et al. Pathological characteristics in retractile testis comparing cryptorchid testis. Korean J Urol. 1999;40:617–22. Mathers MJ, Sperling H, Rubben H, Roth S.  The undescended testis: diagnosis, treatment and long-term consequences. Dtsch Arztebl Int. 2009;106:527–32. Mikuz G.  Update on the pathology of testicular tumors. Anal Quant Cytopathol Histpathol. 2015;37:75–85. Mollaeian M, Mehrabi V, Elahi B.  Significance of epididymal and ductal anomalies associated with undescended testis: study in 652 cases. Urology. 1994;43:857–60.

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Chapter 7

Basic Research Applied to Testicular Torsion Luciano Alves Favorito, Diogo B. de Souza, Daniel Hampl, Carina T. Ribeiro, Marco A. Pereira-Sampaio, and Francisco Jose B. Sampaio

7.1  Introduction Testicular torsion (TT) is a urologic urgency. The testis will present irreversible damage if the torsion is not resolved within 6 hours. Testicular torsion can occur at any age; however, it is more frequent in teenagers and young adults (DaJusta et al. 2013). This pathology is responsible for approximately 90% of acute testicular pain in patients between 13 and 21 years old (Cummings et al. 2002). In this chapter, we show some important aspects about the anatomy and basic research of testicular torsion.

L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil D. Hampl · C. T. Ribeiro Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil D. B. de Souza · F. J. B. Sampaio Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq –Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil e-mail: [email protected] M. A. Pereira-Sampaio Department of Morphology, Fluminense Federal University, Niterói, RJ, Brazil

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_7

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7.2  Embryology of Testicular Torsion and Extravaginal Torsion In humans, about time of male sexual differentiation (7–8  weeks’ gestation) the mesonephros regresses leaving the developing testis, now called mesorchium, on a mesentery. The testicular descent from abdomen to scrotum is a prenatally process and can be divided into two different stages: the transabdominal and inguinoscrotal phases that occur between 10 and 15 weeks and between 25 and 35 weeks of gestation, respectively (Hutson et al. 2013). In male, during the abdominal phase, the cranial suspensory ligament regresses and the gubernaculum enlarges. Once the gubernaculum is attached to the abdominal wall, the testis remains close to the inguinal canal. During the inguinoscrotal phase, the gubernaculum then becomes an elongating structure and migrates to the scrotum. Once reaching the scrotum, the gubernaculum suffers important structural changes and fibrous connection to the surround tissue (Das and Singer 1990). At this time there is also an obliteration of the tunica vaginalis (Favorito et al. 2005) (Fig. 7.1). During that period, because of the mobility of the gubernaculum, extravaginal testicular torsion is likely to happen (Parker and Robison 1971). Failure to this descent process can result in many anatomical aberrations: undescended testis (cryptorchidism), proximal vaginal closure failure (hydrocele, hernia), distal vaginal closure failure (bell clapper deformity), and epididymal disjunctions (long mesorchium) are some examples (Caesar and Kaplan 1994).

Fig. 7.1  Schematic drawing showing the obliteration of processus vaginalis during testicular migration. In the left, the processus vaginalis is opened and in the right it is closed. (Reprinted with permission from the paper of Favorito et al. 2005)

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7.3  A  natomy of Testicular Torsion: Bell Clapper and Intravaginal Torsion In adults, testes are whitish, oval-shaped organs measuring 4.5–5.1 cm and with a volume of approximately 15–25  m (Tishler 1971). Both testicles, once normally positioned inside the scrotum, receive blood and nerve supplies arising from the abdomen through the inguinal cord. Testicular arterial supply and venous drainage are complex and diverse and anomalies are frequent. Multiple or even absence of testicular artery was reported (MacLennan and Hinman 2012). In most cases testicular arteries originate from the abdominal aorta at the level of the second and third lumbar vertebra, just below the origin of the renal artery. Testis and epididymis are irrigated by three main sources: testicular artery (internal spermatic), deferential artery, and cremasteric artery (external spermatic) (Harrison and Barclay 1948). Testis, epididymis, and vas deferens are usually drained by a network of veins that also drains the scrotum and can be divided into superficial and deep. The deep network has an anterior component known as pampiniform complex that fuses into one single vein as it runs into abdomen. The right testicular vein drains into the inferior vena cava and the left into the left renal vein. Lymphatic vessels are prominent along the spermatic cord. The testis has no somatic innervation. They receive their autonomic innervation from the intermesenteric nerve and renal plexus (Baumgarten et al. 1968). The testicular parenchyma is divided into compartments by septa. Each septum divides the seminiferous tubules into compartments and each compartment has a centrifugal artery. Interstitial tissue consists of Leydig cells, mast cells, macrophages, nerves, blood, and lymphatic vessels. In humans, 20–30% of the entire testicular volume correspond to interstitial tissue (Watson et al. 2015) The seminiferous tubules consist of germ cells and Sertoli cells forming a sanctuary – the only environment conducive to the production of male gametes. Sertoli cells are tightly joined and form the most efficient intercellular barrier in the human body. This cellular barrier is the basis of the blood-testicular barrier that allows a privileged immune environment for spermatogenesis (MacLennan and Hinman 2012). Covering and protecting this delicate stroma there is a fibrous membrane called tunica albuginea composed of dense and almost inelastic connective tissue. These anatomical and histological features make testis an extremely sensitive organ to aggressions such as trauma, inflammatory conditions, and ischemia. The testicular torsion is an anomaly resulting from changes in the implantation of the tunica vaginalis or epididymal disjunction (Parker and Robison 1971). Normally, the testis is united to the tunica vaginalis, and if the tunica is implanted too high, the testis can present excessive mobility (bell clapper testis) (Parker and Robison 1971). Mesorchium is the ligament that unites the testis to the epididymis (Scorer and Farrington 1971). In cases of epididymal disjunction or elongated epididymis, conditions that are highly frequent in cryptorchism (Elder 1992; Gill et al. 1989; Gill

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and Kogan 1997; Marshall 1982), the mesorchium is long and can contribute to the testicular torsion (Scorer and Farrington 1971). Testicular torsion is classified into three groups according to the anatomy of the tunica vaginalis and the relationship between testis and epididymis (Favorito et al. 2004) (Fig. 7.2): group A, bell clapper testicular deformity (leading to intravaginal torsion); group B, torsion of spermatic cord (leading to extravaginal torsion); and group C, torsion due to long mesorchium (Fig. 7.2). Testes present a normal layering of tunica vaginalis. They are involved by this structure on both sides and on their upper portion. The posterior region of testis is not covered by tunica vaginalis. United to the lower pole region of the testis and the epididymal tail, there is the testicular gubernaculum or its remnant, the testicular ligament, which is covered by tunica vaginalis only in its anterior and lateral portions (Scorer and Farrington 1971). Intravaginal testicular torsion does not occur in testes presenting normal anatomy as described above, because the posterior testicular segment is firmly united to the scrotum, preventing the organ to move (Parker and Robison 1971; Scorer and Farrington 1971). In a previous study about Testicular torsion (TT) the tunica vaginalis (Favorito et al. 2004) with 25 patients (50 testes and epididymis) with TT in patients were aged between 12 and 23 years (mean 15.6) and torsion duration ranged from 2 hours to 2 days (mean 8 hours) we studied the anatomy of the tunica vaginalis and the

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Fig. 7.2  Testicular torsion is classified into three groups according to the anatomy of the tunica vaginalis and the relationship between testis and epididymis (Fig. 7.1): group A, bell clapper testicular deformity (leading to intravaginal torsion); group B, torsion of spermatic cord (leading to extravaginal torsion); and group C, torsion due to long mesorchium. (Reprinted with permission from the paper of Favorito et al. 2004)

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relationships between testis and epididymis during surgical exploration we observed that 13 (52%) presented torsion of the right testis and 12 (48%) of the left one, with no significant difference between the side of torsion. In eight cases we observed a long mesorchium (three testes (37.5%) were cryptorchid) (Fig. 7.3). Based on our findings, a normal anatomy of tunica vaginalis or epididymis at the side Fig. 7.3 (a) Long mesorchium: a 4-year-old patient with the testis in inguinal position. We can observe a disjunction anomaly between the testis and epididymis and the long mesorchium. (b) A 12-year-old patient with normal insertion of epididymis and testicular torsion by a bell clapper deformity

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contralateral to the torsion is rare (2 cases – 4%), and anatomic anomalies occur bilaterally in the vast majority of cases. These findings stress the need for bilateral orchiopexy in cases of TT. Bell clapper deformity (intravaginal torsion) was the most commonly found type of anomaly (80%). The relation between the presence of full covering of testis and spermatic cord by tunica vaginalis (bell clapper deformity) and testicular torsion is well known. Cases of torsion due to long mesorchium most often occur as a consequence of anomalies of epididymal disjunction or elongated epididymis, conditions that are highly frequent in cryptorchism (Scorer and Farrington 1971; Elder 1992). Of the eight cases with long mesorchium, three (37.5%) had cryptorchid testes. Approximately 20% of cases of testicular torsion occur in patients with cryptorchism (Scorer and Farrington 1971). Epididymal anomalies associated with long mesorchium are frequent in patients with cryptorchism, with an incidence ranging from 36% to 72% (Elder 1992) and rare in individuals with topic testes (less than 4%) (Turek et al. 1994). Due to these changes in mesorchial region, the possibility of testicular torsion must be considered in cryptorchid patients presenting acute scrotal or inguinal pain. The intravaginal torsion (bell clapper tunica vaginalis) is the most frequent type of torsion, and torsion due to long mesorchium is associated with cryptorchism. The most frequently found anatomical relation between testis and epididymis in the study group was type I (epididymis united to the testis by its head and tail). Historically the bell clapper deformity is the only anatomical risk factor for the intravaginal torsion, but the epididymal disjunction, despite rare, also plays a hole in the pathology. In approximate 90% of the time, a vestigial of Müllerian duct keeps present attached to the upper pole of the testis and is called appendix testis or hydatid of Morgagni. This appendix can also twist during lifetime, mimics the testicular torsion clinical presentation, and sometimes also requires surgical treatment (Sakurai et al. 1983; Noske et al. 1998) (Fig. 7.4).

Fig. 7.4  A vestigial of Müllerian duct keeps present attached to the upper pole of the testis and is called appendix testis or hydatid of Morgagni in a patient with 5 years old and the right testis in inguinal position

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7.4  Clinical Aspects of Testicular Torsion Testicular torsion is an ischemic emergency. In the setting, testicular functional tissue is damaged in many pathways, which leads to hypoxia and cellular death and testicular atrophy. Signs and symptoms of torsion include acute scrotal pain and testicular ascent, and the testis can lie horizontally with or without inflammatory signs (Ben-Chaim et  al. 1992; Kass et  al. 1993). The diagnosis is made mainly through clinical examination; however, in doubtful cases Doppler ultrasonography of the spermatic cord and testicular scintigraphy can be used to assess testicular perfusion (Middleton et al. 1990). Often these tests are not promptly available; thus in doubtful cases following clinical examination, when complementary exams cannot be performed, urgency scrotal exploration is the treatment of choice (Kass et al. 1993) (Fig. 7.5). The treatment of TT is the repositioning and fixation of affected testicles (if it is still considered viable) or orchiectomy (if it is considered inviable). In spite of the condition of the twisted testicle, it is recommended to perform orchiopexy in the contralateral organ, to avoid future TT (Bolln et al. 2006). The goal of orchiopexy is to maintain the testicle in its correct position, with preservation of vascular supply and as the prevention of testicular torsion recurrence (Lotan et al. 2005). Although this surgery is intended to prevent the loss of testicular function, testicular damage has been associated with orchiopexy. Orchiopexy is thought to lead to testicular damage by different mechanisms. One theory is that the tied knot on the testicular parenchyma acts as a ligature, leading to ischemia of part of the organ. Thus, the number and site of the knots have been addressed by some authors (Coughlin et al. 1998; Jarrow 1991). Other authors have shown that the type of suture influences the morphological and functional outcomes following orchiopexy. This would lead to the production of autologous antisperm antibodies, leading to damage to the ipsilateral and contralateral testicle (Bellinger et al. 1989). a

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Fig. 7.5  A 14-year-old patient with testicular torsion for more than 12 hours. (a) Clinical exam showed the right testis in high position and (b) the typical aspect of necrosis in this case was observed during the surgery

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Spermatic cord torsion increases first the pressure of the venous drainage; in other words the venous return is compromised. An edema increases pressure inside the testicle and, because of the inelastic characteristic of the tunica albuginea, a compartment syndrome is installed. Both the arterial supply torsion and compartment syndrome nullify the capacity of tissue irrigation that, not forgetting, is performed by terminal arterioles (Kutikov et al. 2008) Time from symptoms to the surgical intervention is the main predictor of testicular salvage and prevention of late testicular atrophy. Unfortunately, even untwisting the spermatic cord might also promote testicular injure because an ischaemia-­ reperfusion injury – specially, considering the brake of the blood-testicular barrier (Kutikov et al. 2008). Several clinical and experimental studies on testicular torsion are described in the literature (Ben-Chaim et al. 1992; Kass et al. 1993). Studies on the anatomic aspects of the tunica vaginalis and the association with epididymal anatomy and its anomalies in patients with testicular torsion are scarce (Caesar and Kaplan 1994). Testicular torsion occurs due to anatomic anomalies of tunica vaginalis or epididymis that allow excessive testicular mobility inside the scrotum. Due to this excessive mobility, testis can present medial rotation that ranges from 360° to 720° in its own axis, which can cause interruption of the organ’s vascularization (Ben-­ Chaim et al. 1992).

7.5  A  ge of Onset and Its Influence on Function and Morphology of the Adult Testicle Applied to Testicular Torsion To investigate the impact of the age of TT occurrence we assessed the testicular morphology, spermatozoid parameters, and reproductive function of 58 male Wistar rats (Ribeiro et al. 2014). Unrelated females (three females per male) were used for the fertility tests as described below. Three age groups were used: prepuberty, puberty, and adulthood (4, 6, and 9 weeks of age, respectively). Each age group was composed by a sham-operated group and a group submitted to testicular torsion (TT). This resulted in six experimental groups: SH4, n = 10 (prepubertal animals submitted to simulated surgery); TT4, n  =  10 (prepubertal animals submitted to testicular torsion); SH6, n  =  10 (pubertal animals submitted to simulated surgery); TT6, n  =  9 (pubertal animals submitted to testicular torsion); SH9, n = 10 (adult animals submitted to simulated surgery); and TT9, n = 9 (adult animals submitted to testicular torsion). After general anesthesia induction, TT was induced by opening the scrotum and the lamina parietalis of the tunica vaginalis and twisting the right testicle 720° clockwise (Fig. 7.6). The twisted testicle was fixed in position by sutures and torsion maintained for 4 hours while under general anesthesia. After this period, the organs were untwisted and fixed in anatomic position. Sham animals had the same

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Fig. 7.6  Testicular torsion experimental model. (a) Image of a right testicle before torsion in a rat model; (b) image of a right testicle after 4 hours of 720° torsion and immediately before detorsion

surgical approach to open the tunica vaginalis. The testicle was sutured in anatomical position for the same period but not twisted. At 12 weeks of age, all male rats were mated with three estrous females to obtain fertility parameters (Motrich et al. 2007). Females were euthanized on day 20 of gestation. The uterus was opened, pregnancy was confirmed, and the number of fetuses and implantation sites were recorded. The ovaries were observed under magnification and the number of corpora lutea was counted. Potency was calculated as the percentage of female rats with confirmed copula over the number of female rats exposed for mating. Fertility index was calculated as the percentage of implantation sites over the number of corpora lutea. The fecundity of each group was considered the percentage of male rats that generated at least one fetus over the total number of male rats of the group. Also, preimplantation and postimplantation losses were calculated. All males were euthanized at 14 weeks of age by anesthetic overdose. Just after death, spermatozoids were collected from epididymal tail to determine the concentration and motility in a Neubauer chamber. Also, spermatozoid viability was assessed by the hypoosmotic test. For these analyses, 200 spermatozoids were evaluated per animal. Since samples were collected and analyzed from both right (twisted) and left (contralateral) epididymis, the letter R or L (right or left) was added to the group name to designate the origin of the sample (e.g., SH4R and TT10L). The same denominations were used to describe morphological analysis. After euthanasia, both testicles were dissected from their appendices and weighed and their volume was measured using the Scherle’s method (de Souza et al. 2011). Subsequently the organ was fixed and processed for paraffin embedding for obtaining 5-μm-thick histological sections. Morphometric analysis was performed on hematoxylin and eosin-stained slices and captured on an Olympus BX51 microscope with a coupled DP70 digital camera (Olympus, Tokyo, Japan). The volumetric density (Vv) of testicular structures was assessed by the point counting method. Using the ImageJ software (NIH, Bethesda, Maryland, USA), a test grid with 100 points was superimposed over the testicular photomicrographs.

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Every point-touched structure was counted and its density was expressed in percentage of the analyzed field. For each testicle, 25 fields were evaluated under 400× magnification. The Vv of the following structures were recorded: tunica propria, seminiferous epithelium, tubular lumen, seminiferous tubule (the sum of these first three structures), vessels, and the intertubular compartment (including vessels). The absolute volume (Av) of each of the abovementioned structures, expressed in milliliters, was calculated by dividing the testicular volume by the Vv of the structure and total tubular length was calculated as previously described (Silva et al. 2010). The diameter of 125 seminiferous tubules per animal was measured in each testis by applying a straight line crossing the tubule. For this purpose we used the ImageJ software, previously calibrated for the magnification of 100×. The line was applied in a manner that it always passes through the center of the tubule. For this analysis tubules with irregular shapes were excluded. Also with this software, the seminiferous epithelium height of randomly picked tubules was measured in each testis. In this analysis, 125 tubules per animal were assessed in images photographed at 200× magnification. Cellular proliferation was assessed in the interstitial and tubular spaces separately. Histological sections were immunostained with proliferation cell nuclear antigen (PCNA) antibodies (180,110, Invitrogen, Camarillo, EUA) with Histostain Plus labeling (859,643, Invitrogen, Camarillo, EUA). These sections were photographed under 400× magnification. The number of positive cells per mm2 of interstitium and seminiferous tubules was quantified using the ImageJ software. The results of groups TT4, TT6, and TT9 were compared to investigate what part age of onset of TT might have in adult testicular function and morphology. Regarding the investigated parameters of the fertility test, no statistical difference was found among the groups. Concerning spermatozoid analysis, all rats with TT had lower concentration, motility, and viability than the sham groups. Samples from TT9R were completely lacking of spermatozoid. Even so, the statistical analysis of spermatozoid concentration did not point to difference among the TT groups. With no spermatozoid in TT9R, motility and viability analysis were performed only on TT4R and TT6R samples with no statistical difference between them. Also, testicular volume and weight were not different among the TT4R, TT6R, and TT9R groups (Fig. 7.7). However, histological parameters, Av and Vv [seminiferous epithelium] of TT6R, were higher than TT4R and TT9R, indicating that testicular torsion occurring during puberty may be less harmful to testicular morphology than when it occurs before puberty or at adult age as noted in Fig. 7.7. From these results one can suggest that more effort should be given to adult patients presenting TT, in order to diminish the time from the beginning of ischemia to surgical resolution. Also, more research about adjuvant therapies that could ameliorate testicular damage would be of special importance for use in adult patients. Spermatozoid parameters were not statistically improved by antioxidant treatment. However in rats submitted to TT at adulthood both drugs aided recovery of some viable spermatozoid in the twisted testis, which seems promising for clinical use.

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Fig. 7.7  Right and left testicles of an animal from the group of pubertal animals submitted to testicular torsion. The drastic reduction of volume in testicles submitted to torsion can be easily noted on visual inspection

With the obtained results on this study we concluded that the age of TT occurrence does not influence spermatozoid production or fertility in adulthood. However, the testicular morphology is less affected in animals submitted to TT during puberty.

7.6  Influence of Antioxidant Treatment in Testicular Torsion To investigate the effects of antioxidant administration during the TT perioperative period we used 106 male Wistar rats. Unrelated females (three females per male, n = 318) were used for the fertility tests as described below. The male animals were divided into 12 groups according to age (prepuberty, puberty, and adulthood, with 4, 6, and 9 weeks of age, respectively) and treatment (Sham, TT without antioxidant therapy, TT with resveratrol treatment (RES), and TT with arginine treatment (ARG)). This resulted in the following experimental groups: SH4, n  =  10 (prepubertal animals submitted to simulated surgery); TT4, n = 10 (prepubertal animals submitted to TT without antioxidant treatment); RES4, n  =  8 (prepubertal animals submitted to TT with resveratrol treatment); ARG4, n = 8 (prepubertal animals submitted to TT with arginine treatment); SH6, n = 10 (pubertal animals submitted to simulated surgery); TT6, n  =  9 (pubertal animals submitted to TT without antioxidant treatment); RES6, n = 8 (pubertal animals submitted to TT with resveratrol treatment); ARG6, n = 8 (pubertal animals submitted to TT with arginine treatment); SH10, n = 10 (adult animals submitted to simulated surgery); TT10, n  =  9 (adult animals submitted to TT without antioxidant treatment); RES10, n = 8 (adult animals submitted to TT with resveratrol treatment); and ARG10, n = 8 (adult animals submitted to TT with arginine treatment).

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Groups RES4, RES6, and RES10 received resveratrol (30 mg/kg) intraperitoneally, 30 minutes before testicle detorsion. For 7 days post-operatory, resveratrol was administered daily by gavage at the same dose (Uguralp et al. 2005). Groups ARG4, ARG6, and ARG10 received arginine (650  mg/kg) by gavage for 7  days post-operatory. All analyses performed in the previous experiment (spermatozoid analysis, fertility tests, and testicular morphological studies) were also used in this study. The results of the Sham, TT, RES, and ARG groups were compared to verify if antioxidant treatment could prevent the testicular damage. Fertility tests demonstrated some improvement in the groups treated with antioxidants. Rats from RES4 and RES6 showed an increased potency of 52% and 142%, respectively, in comparison to untreated animals. Animals from groups ARG4, ARG6, and ARG10 also had an increase of 66%, 187%, and 113% in this parameter. Interestingly, animals from ARG6 had a potency even higher than animals submitted to sham surgery at the same age. Both antioxidants also improved fecundity in animals submitted to TT at 9 weeks old. TT induced a marked decrease in spermatozoid production and quality in twisted testicles, regardless of the age of occurrence. The spermatic damage was not prevented by antioxidant treatment. Spermatozoids viability of RES6R was the only improvement noted regarding antioxidant treatment. In this group, the results showed a partial protection, since it was not statistically different from SH6R and from TT6R.  Interestingly, both antioxidants improved contralateral spermatozoid quantity and quality (Fig. 7.8). Regarding testicular weight and volume, TT induced a great decrease (46–64%) regardless of the age of occurrence. Resveratrol treatment prevented this decrease in the 4-week-old group, while arginine partially protected atrophy in animals submitted to TT at 6 and 9 weeks old. Resveratrol prevented changes of the Av [tubular lumen], Vv [blood vessels], and total tubular length in RES4R (Fig. 7.9). In RES9R, resveratrol prevented cellular proliferation in the interstitial space. It was also partially effective in preventing changes in Av [tubular compartment], Vv [intertubular compartment], and tubular cellular proliferation in RES4R samples. In RES6R, resveratrol partially prevented changes of the Vv [intertubular compartment]. In RES9R, resveratrol partially prevented the reduction of epithelial height. However, resveratrol had a negative effect in some parameters, lowering the Vv [tunica propria] in testicles twisted at 4 and 6  weeks old and decreasing the Vv [tubular compartment] in testicles twisted at 6 weeks old. Arginine was partially effective to prevent changes in seminiferous tubule diameter, seminiferous epithelium height, Vv [seminiferous epithelium], Vv [blood vessels], Av [seminiferous epithelium], Av [tunica propria], and cellular proliferation of tubular compartment in ARG4R. In ARG9R, arginine prevented the raise of the cellular proliferation in the interstitial space. Also, in ARG9R it partially prevented the reduction of epithelial height. However, arginine had a negative effect in some

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Fig. 7.8  Histological images of the testis in rats of the sham group and testicular torsion group (TT) without antioxidant therapy. The figures show the right testicle seminiferous tubular damage in prepubertal, pubertal, and adult rats. H&E ×200. TT induced a marked decrease in spermatozoid production and quality in twisted testicles, regardless of the age of occurrence. The spermatic damage was not prevented by antioxidant treatment. (a) SH4, sham group without antioxidant therapy in prepubertal animals submitted to simulated surgery; (b) TT4, prepubertal animals submitted to TT without antioxidant treatment; (c) SH6, sham group in pubertal animals submitted to simulated surgery; (d) TT6, pubertal animals submitted to TT without antioxidant treatment; (e) SH10, adult animals submitted to simulated surgery; and (f) TT10, adult animals submitted to TT without antioxidant treatment

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Fig. 7.9  Histological images of the testis in rats treated with arginine or resveratrol. H&E ×200. Resveratrol prevented changes of the tubular lumen, blood vessels, and total tubular length. The figures show right testicle seminiferous tubular damage in prepubertal, pubertal, and adult rats. (a) ARG4, prepubertal animals submitted to TT with arginine treatment; (b) RES4, prepubertal animals submitted to TT with resveratrol treatment; (c) ARG6, pubertal animals submitted to TT with arginine treatment; (d) RES6, pubertal animals submitted to TT with resveratrol treatment; (e) ARG10, adult animals submitted to TT with arginine treatment; and (f) RES10, adult animals submitted to TT with resveratrol treatment

parameters, lowering the Vv [tunica propria] and Av [blood vessels] in ARG4R, reducing the Vv and Av [seminiferous epithelium] in ARG6R, and increasing Vv [blood vessels] in ARG9R. This study evaluated for the first time the effect of treatment with two different antioxidants on morphological, spermatic, and fertility parameters after TT occurring in different ages. Animals submitted to TT before puberty were benefited most by antioxidants, with a significant protection of tubular structures. In contrast, the

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treatments were of little help to animals operated during puberty. It is difficult to explain these findings, but possibly the pubertal testicle would be more tolerant to disruption while prepubertal testicles would be more susceptible to oxidative stress and, consequently, more protectable by antioxidants. Regardless of the mechanisms on how these antioxidants act in TT, its usage could help to preserve testicular function and this may have a more prominent effect in infantile patients. From this study we concluded that the treatment with resveratrol or arginine ameliorated testicular morphology after TT.  Although the antioxidants did not enhance spermatozoid parameters in the twisted testicles, it improved contralateral testicle spermatozoid production and some fertility parameters. Prepubertal animals were the most benefited of both antioxidant treatments.

7.7  Influence of Orchiopexy in Testicular Torsion To investigate the testicular alterations after orchiopexy (transparenchymal suturing) 40 male Wistar rats were used. The rats were randomly assigned to four groups, with ten animals in each group. The control group was constituted of animals of 14 weeks of age, kept under normal conditions, and not submitted to any procedure until euthanasia. The other three experimental groups were divided according to the animal’s age; the experiment used prepubertal, pubertal, and adult rats at 4, 6, and 9 weeks of age, respectively. The animals of these three experimental groups were submitted to the surgical procedures described below. Under anesthesia with ketamine (80 mg/kg) associated with xylazine (10 mg/kg) and aseptic techniques, the right testicle was exposed by opening the scrotum and tunica vaginalis. Then, the organ was penetrated in its two margins (lateral and medial) with a polyglactin 910 suture, without tying the knots (Fig. 7.10). For prepubertal and pubertal animals, 6-0 sutures were used, while 4-0 sutures were chosen for adult rats. Suture caliber was selected according to the thickness of the tunica albuginea as the suture used should not be thicker than this tunic. The sutures were applied by penetrating the tunica albuginea and the testicular parenchyma at the middle third of the lateral and medial margins of the organ, avoiding the visible vasculature. The scrotum was closed and the testicle retained with the sutures for 4 hours, while the animals were maintained under general anesthesia. After this period, the scrotum was reopened, the sutures applied were removed, and the scrotum was definitely closed. The contralateral (left) testicles remained untouched and were used for comparison to the right testicles. Thus, for the proposed analysis, this experiment had seven different groups: Ctrl, samples from the right testicles/epididymis of control animals; PrCl, samples from the contralateral (left) testicles of animals operated on during the prepubertal age; PrSu, samples from sutured (right) testicles of animals operated on during the prepubertal age; PuCl, samples from contralateral testicles of animals operated on during puberty; PuSu, samples from sutured testicles of animals operated on during puberty; AdCl, samples from the contralateral testicles

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Fig. 7.10  Testicle in a rat model with suture applied at the lateral and medial margins

of adult operated animals; and AdSu, samples from sutured testicles of adult operated animals. All animals were submitted to euthanasia within 14 weeks of age by anesthetic overdose and samples were collected for sperm and testicular morphological analysis as previously described in the other studies. For each parameter analyzed, the means of right and left testicles of operated animals in each age group (prepubertal, pubertal, and adult) were compared to the right testicle of the control group. Regarding the sperm concentration, no differences were observed among the samples collected from the tail of the epididymis of controls and contralateral and sutured organs, regardless of the age of the animal when the suture was applied. In addition, sperm motility was similar among controls and contralateral and sutured organs in animals submitted to parenchymal suture before or during puberty. However, animals operated on at adult life showed a 29.5% decrease sperm motility in samples collected from the tail of the epididymis of sutured organs, compared to samples collected from controls. The hyposmotic test showed a decrease in sperm viability of 28.4% in contralateral samples, as well as a reduction of 32.5% in samples collected from the tail of the epididymis of sutured organs in adult operated animals compared to controls. Even so, in prepubertal and pubertal operated animals no difference was found. In prepubertal operated animals, the seminiferous tubule diameters of contralateral and sutured testicles were reduced by 10% and 12% compared to controls, respectively. This difference was not observed in animals operated on during pubertal or at adult life. Also, no difference was observed in seminiferous epithelium height among the groups, for both ages.

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The analysis of tunica propria volumetric density indicates that this structure is augmented in both sutured and contralateral testicles, in comparison to controls. This was observed in prepubertal, pubertal, and adult operated rats. In prepubertal operated animals, sutured testicles showed an 85% increase in this parameter while contralateral testicles showed a twofold increase, in comparison to controls. In rats submitted to surgery during puberty, the contralateral testicles had a 57% increase and sutured organs presented an 85% increase in tunica propria volumetric density, when compared to controls. In adult operated animals, the tunica propria of the sutured and contralateral testicles was increased by 44% and 50% compared to controls The seminiferous epithelium volumetric density was also altered by parenchymal suture. In prepubertal operated animals, the contralateral testicle had a 20% reduction in comparison to controls. In sutured organs of these animals, a 35% reduction was observed. For animals operated during puberty, the sutured organs showed a 10% decrease in this parameter. Contralateral testicles did not present any differences in comparison to controls. In rats submitted to testicular suture in adult life, the seminiferous epithelium volumetric density was reduced by 23% in both sutured and contralateral testicles, when compared to controls. Although the volumetric density of luminal space did not show differences among all analyzed animals, the tubular compartment volumetric density (which is calculated as the sum of tunica propria, seminiferous epithelium, and luminal space volumetric densities) was altered in animals operated before and after puberty. In prepubertal operated animals, the tubular compartment was reduced by 20% in sutured animals, in comparison to controls. In adult operated rats, this compartment was reduced by 5% in sutured organs, in comparison to controls. In animals operated on during puberty, no difference was found. Finally, differences in total seminiferous tubule length were observed only among adult operated animals. The testicles of these animals that received sutures presented a 14% decrease in comparison to controls. The results clearly demonstrated that only the mechanical parenchymal transfixation of the suture, with no knot, causes enough damage to promote morphological alterations in the testes. In this study we show that some morphological changes found in the operated testis were also found in the contralateral testis, and this occurred when orchiopexy was performed at the three different ages studied, being more intense in the prepubertal group. This could be an effect of free radicals or could be only an effect of the anesthetic procedure. Ketamine, one of the anesthetics used in the present study, is known to reduce LH levels during the first 2 hours after systemic administration. Although the objective of the study was to evaluate the transparenchymal suture as a single factor, it should be pointed out that this is not the common setting in clinical practice where other concomitant factors may contribute to the final reproductive function in patients. Also, another limitation of this study is that fertility parameters were not assessed. Further studies evaluating the effects of transparenchymal suture on fertility at adult ages are of interest. From the results obtained we can conclude that the transfixation of the rat testicular parenchyma is sufficient to cause important morphologic damage to the

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operated testicle, as well as to the contralateral one. Such changes are seen at different ages (before, during, and after puberty); however, in individuals operated during puberty, these changes seem to be less severe. Thus, the use of other techniques for orchiopexy, not involving parenchyma transfixation, should be preferred, regardless of the age at which this procedure is performed.

References Baumgarten HG, Falck B, Holstein AF, et  al. Adrenergic innervation of the human testis, epididymis, ductus deferens and prostate: a fluorescence microscopic and fluorimetric study. Z Zellforsch Mikrosk Anat. 1968;90:81–95. Bellinger MF, Abromowitz H, Brantley S, et al. Orchiopexy: an experimental study of the effect of surgical technique on testicular histology. J Urol. 1989;142:553. Ben-Chaim J, Leibovitch I, Ramon J, Winberg D, Goldwasser B.  Etiology of acute scrotum at surgical exploration in children, adolescents and adults. Eur Urol. 1992;21:45–7. Bolln C, Driver CP, Youngson GG. Operative management of testicular torsion: current practice within the UK and Ireland. J Pediatr Urol. 2006;2:190. Caesar RE, Kaplan GW. Incidence of the bell-clapper deformity in an autopsy series. Urology. 1994;44:114–6. Coughlin MT, Bellinger MF, LaPorte RE, et al. Testicular suture: a significant risk factor for infertility among formerly cryptorchid men. J Pediatr Surg. 1998;33:1790. Cummings JM, Boullier JA, Sekhon D, Bose K. Adult testicular torsion. J Urol. 2002;167:2109–10. DaJusta DG, Granberg CF, Villanueva C, Baker LA. Contemporary review of testicular torsion: new concepts, emerging technologies and potential therapeutics. J Pediatr Urol. 2013;9(6, Pt. A):723–30. Das S, Singer A. Controversies of perinatal torsion of the spermatic cord: a review, survey and recommendations. J Urol. 1990;143:231–3. de Souza DB, Silva D, Marinho CSC, et al. Effects of immobilization stress on kidneys of Wistar male rats: a morphometrical and stereological analysis. Kidney Blood Press Res. 2011;34:424. Elder JS.  Epididymal anomalies associated with hydrocele/hernia and cryptorchidism: implications regarding testicular descent. J Urol. 1992;148:624–6. Favorito LA, Cavalcante AG, Costa WS. Anatomic aspects of epididymis and tunica vaginalis in patients with testicular torsion. Int Braz J Urol. 2004;30(5):420–4. Favorito LA, Costa WS, Sampaio FJ. Relationship between the persistence of the processus vaginalis and age in patients with cryptorchidism. Int Braz J Urol. 2005;31(1):57–61. Gill B, Kogan S. Cryptorchidism. Current concepts. Pediatr Clin North Am. 1997;44:1211–27. Gill B, Kogan S, Starr S, Reda E, Levitt S. Significance of epididymal and ductal anomalies associated with testicular maldescent. J Urol. 1989;142:556–8; discussion 572. Harrison RG, Barclay AE. The distribution of the testicular artery (internal spermatic artery) to the human testis. Br J Urol. 1948;20:5. Hutson JM, Southwell BR, Li R, Lie G, Ismail K, Harisis G, Chen N.  The regulation of testicular descent and the effects of cryptorchidism. Endocr Rev. 2013;34(5):725–52. https://doi. org/10.1210/er.2012-1089. Jarrow JP. Clinical significance of intratesticular anatomy. J Urol. 1991;145:777. Kass EJ, Stone KT, Cacciarelli AA, Mitchell B.  Do all children with an acute scrotum require exploration? J Urol. 1993;150:667–9.

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Kutikov A, Casale P, White MA, Meyer WA, Chang A, Gosalbez R, Canning DA.  Testicular compartment syndrome: a new approach to conceptualizing and managing testicular torsion. Urology. 2008;72(4):786–9. Lotan G, Golan R, Efrati Y, et al. An experimental study of the effect of two distinct surgical techniques of orchiopexy on spermatogenesis and testicular damage in cryptorchid testes. Fertil Steril. 2005;84:749. MacLennan GT, Hinman F. Hinman’s atlas of urosurgical anatomy. 2nd ed. Philadelphia: Elsevier/ Saunders; 2012. p. 368. Marshall FF. Anomalies associated with cryptorchidism. Urol Clin North Am. 1982;9:339–47. Middleton WD, Siegel BA, Melson GL, Yates CK, Andriole GL. Acute scrotal disorders: prospective comparison of color Doppler US and testicular scintigraphy. Radiology. 1990;177:177–81. Motrich RD, Ponce AA, Rivero VE. Effect of tamoxifen treatment on the semen quality and fertility of the male rat. Fertil Steril. 2007;88:452. Noske HD, Kraus SW, Altinkilic BM, Weidner W. Historical milestones regarding torsion of the scrotal organs. J Urol. 1998;159:13–6. Parker RM, Robison JR. Anatomy and diagnosis of torsion of the testicle. J Urol. 1971;106:243–7. Ribeiro CT, Milhomem R, De Souza DB, et al. Effect of antioxidants on outcome of testicular torsion in rats of different ages. J Urol. 2014;191:1578. Sakurai H, Ogawa H, Higaki Y, Yoshida H, Imamura K. Torsion of appendix of testis and epididymis: a report of 4 cases. Hinyokika Kiyo. 1983;29:1657–68. Scorer CG, Farrington GH.  Congenital deformities of the testis and epididymis. London: Butterworths e Co; 1971. Silva RC, Costa GM, Andrade LM, et al. Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis). Theriogenology. 2010;73:157. Tishler PV. Diameter of testicles. N Engl J Med. 1971;285:1489. Turek PJ, Ewalt DH, Snyder HM 3rd, Duckett JW. Normal epididymal anatomy in boys. J Urol. 1994;151:726–7. Uguralp S, Mizrak B, Bay Karabulut A.  Resveratrol reduces ischemia reperfusion injury after experimental testicular torsion. Eur J Pediatr Surg. 2005;15:114. Watson MJ, Bartkowski DP, Nelson NC. Intracompartmental pressure as a predictor of intratesticular blood flow: a rat model. J Urol. 2015;193:2062–7.

Chapter 8

Methods of Basic Research Applied to Urinary and Genital Systems During the Human Fetal Period Luciano Alves Favorito and Francisco Jose B. Sampaio

8.1  Fetal Measurements The first step of fetal investigation applied to translational research in pediatric urology is the determination of fetal age. We describe all the steps to determine the fetal measurements and fetal age. The fetuses studied are donations to our laboratory from the obstetric section of our hospital. The fetuses were macroscopically well preserved with grade I classification, according to Streeter (1920): rosy, shiny, firm tissues, and no traumas, hematomas, or congenital malformations (Fig. 8.1). After determination of death, the fetuses are kept in refrigeration (temperature lower than 4 centigrade grades) for 24–72 hours. After reaching the laboratory, the fetuses are defrosted, cleaned, identified, and analyzed morphologically. Fetuses with malformations or not well preserved are excluded for analysis. After cataloguing, the first step is to weigh, using a precision scale of 1  g (Fig. 8.2). The fetuses are also evaluated regarding crown-rump length (CRL), total length (TL), and foot length immediately before dissection (Fig.  8.3). The same observer analyzes all measurements. For the evaluation of the CRL and TL, a metric tape is used, and to check the length of the bigger foot (more posterior region from the heel to the tip of the most prominent toe, first or second), a Starrett® digital pachymeter 0.01 cm precision is used (Fig. 8.4). The measures of the right and left feet are measured three times each, using the millimeter precision pachymeter (mm). The foot with the higher median is used to determine the gestational age, L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil F. J. B. Sampaio Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_8

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Fig. 8.1 Well-preserved fetuses without apparent anomalies. Both are second trimester fetuses. Only fetuses like those are used in our studies

lowering the risk of error (Spencer and Coulombe 1964). That measure is analyzed in a graphic (Streeter 1920) that relates the length of the bigger foot with gestational age, according to weeks after conception (WPC) (Drumm 1976; Mercer et al. 1987; Sampaio and Favorito 1998). The gestational age of the fetuses is determined in WPC, according to the foot length criterion, which is currently considered the most acceptable parameter to calculate gestational age (Streeter 1920; Casey and Carr 1982; Hern 1984; Platt et al. 1988; Favorito and Sampaio 1998). After the fetal measurements, the fetuses are carefully dissected with the aid of a stereoscopic lens with 16/25× magnification (Fig. 8.5). The abdomen and pelvis are opened to identify and expose the urogenital organs (Fig. 8.6) and take the organs to histologic analysis.

8  Methods of Basic Research Applied to Urinary and Genital Systems… Fig. 8.2  Precision scales used in the study. (a) Scale used to check the weight of bigger fetuses; (b) 25 weeks postconception fetus being weighted; (c) analytical precision scale used to weigh fetuses with less than 300 g

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Fig. 8.4 Precision pachymeter to check the measure of the bigger foot – from the most prominent toe to heel. This measure is the most important for the determination of the gestational age

8.2  Histologic Techniques The samples are separated from the other structures and fixed in 10% buffered formalin and routinely processed for paraffin embedding, after which 5-μm-thick sections are obtained at 200 μm intervals. Smooth muscle and connective tissue, elastic system fibers, and collagen are studied by histochemical and immunohistochemical methods.

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Fig. 8.5  The figure shows a stereoscopic lens with 16/25× magnification used to dissect the urogenital organs of the fetuses after fetal measurements

Fig. 8.6  The figure shows the steps of fetal dissection. (a) The abdominal wall of a female fetus aged 25 weeks postconception is opened; (b) the urogenital organs (uterus, bladder, kidneys) are dissected and removed in block for analysis

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Sections are stained with hematoxylin-eosin to assess the integrity of the tissue. The following staining methods are used: Masson’s trichrome, to quantify connective and smooth muscle tissue; Weigert resorcin-fuchsin with previous oxidation, to observe elastic system fibers; and picrosirius red with polarization for observation of different collagen types. The immunohistochemical analysis of gubernaculum nerves is done with tubulin (tubulin, beta III, mouse monoclonal antibody).

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8.2.1  Collagen Fibers (a) Gomori and Masson trichrome (Bancroft and Cook 1994) • Routinely used technique to highlight the collagen component that stains green (Gomori trichrome) or blue (Masson trichrome). The cell cytoplasm, when submitted to this stain, may turn red due to chromotrope 2R, present in the solution. ( b) Picrosirius (under polarized light or not) (Junqueira et al. 1979) • Picrosirius is a strongly acid stain, due to the presence of six sulfonic radicals that react to aminic groups of lysine molecules, one of the main basic amino acids that form the collagen molecule. The ligation parallels the bigger axis with collagen, promoting an increase of their normal birefringence (Montes et al. 1984). Collagens type I, II, and III may present different intensity colors of refringence in the same tissue slice. This fact may be explained due to variations of aggregation pattern of different collagens. In general, thick or strongly aggregated fibers (usually type I collagen) present birefringence from yellow to red tones. Finer fibers (collagen type III) show a more discrete birefringence with a greenish tone. Collagen type II (fibrillar collagen) forms a net intimately associated with amorphous substance, producing a weak birefringence with variable color (Junqueira et al. 1979).

8.2.2  Reticular Fibers (a) Gomori reticulin (Gomori 1937) • This stain technique is used to show fibers traditionally called reticular fibers, composed of type III collagen, which molecules form fibrils that are thin and highly ramified. Collagen fibers type III are associated with the abundant interfibrillar material rich in carbohydrates. • The more efficient methods for their identification are those that use argyrophilia due to the high concentration of glycosidic residues associated with collagen molecules. • The technique is based on the treatment of tissue with a metal solution. Initially, reticular fibers are formed in submicroscopic sites sensitive to silver, in its reduced form, that next is converted to metallic silver (using a reduction agent) that is deposited in sensitive sites (Bradburry and Rae 1996). • Reduction of metallic silver (from the ammoniacal silver solution) depends on reactive groups that are present in the carbohydrate matrix (reduction agents) that forms in response to glycoprotein oxidation associated to collagen type III fibrils. Metallic silver deposited in sensitive sites are observed in dark brown or back colors (Bradburry and Rae 1996).

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• Usually, an oxidation step by potassium permanganate is needed (to eliminate normal argyrophilia from structures such as nervous fibers). The use of gold chloride is recommended to clean the background and consequently ease the visualization of black fibers.

8.2.3  Elastic System Fibers (a) Weigert fuchsin-resorcin, with or without previous oxidation with Oxone (Fulmmer 1958; Cotta-Pereira et al. 1977). • Weigert reagent is formed by the precipitation of basic fuchsin-resorcin by iron chloride, demonstrating the presence of elastic and elauninic fibers, and following an oxidation step with para-acetic/Oxone acid, and also the presence of oxitalanic fibers. The initial step with a solution of acidified potassium permanganate and oxalic acid clears the background (Bancroft and Cook 1994), highlighting the elastic fibers dyed in dark violet.

8.2.4  Muscular Fibers (a) Gomori and Masson’s trichrome • Previously described 8.2.4.1  Immunohistochemistry The widely used immunohistochemistry technique is avidin-biotin technique to identify collagenous proteins, elastin, and glycoproteins (Beesley 1993; True 1990). The following primary polyclonal antibodies are used: (a) Human anti-elastin diluted 1:2 (No. 25011/165, Institut Pasteur) (b) Anti-α-human actin (Sigma Immuno Chemical Product No. A-2547) The antibodies are revealed with the use of a Mouse Rapid Staining Kit (Stock #1, Quik-1 – Sigma Chemical Co., St. Louis, USA), containing a secondary antibody, peroxidase, 3% hydrogen peroxide, and the chromogen AEC (3-amino-9-etil-carbazole). Histological slices are deparaffinized, hydrated in alcohol with different concentrations until water, and then washed in PBS for 5  minutes; next, the slices are washed for 5  minutes with two drops of 3% hydrogen peroxide in methanol, to block the activity of endogenous peroxidase. Next, an enzymatic treatment is performed with trypsin 0.1% in PBS for 30 minutes in a humid chamber at 37 °C with normal goat serum to block the nonspecific connection sites. The material is

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incubated in a humid chamber at 4 °C in a refrigerator, overnight. After washing with PBS, the slices are incubated with a secondary antibody, in a humid chamber at room temperature, for 30  minutes. The activity of peroxidase is shown with a 0.1% AEC solution (Beesley 1993; True 1990). The slices are counterstained with Mayer hematoxylin for 1 minute, mounted in glycerol-gelatin (Sygma Product No. GG-1), analyzed, and photographed in a microscope model Hund H 500. Simultaneously, a negative control is performed for all primary antibodies, and the primary antibody is replaced by PBS, and positive controls use tissue fragments that in literature showed the researched antigens. Therefore, mouse skin for elastin and mouse aorta for α-actin are used.

8.3  Quantification Techniques Connective tissue, smooth muscle tissue, nerves, and elastic system fibers are quantified by a stereological method (Mandarim-de-lacerda 2003; Mandarim-de-Lacerda et al. 2010; Chagas et al. 2002). We study five microscopic fields chosen at random, totaling 25 test areas studied for each gubernaculum for the quantitative analysis. We use the ImageJ software, version 1.46r, loaded with its own plug-in (http://rsb.info. nih.gov/ij/). All sections are photographed with a digital camera (DP70, Olympus America, Inc., Melville, New  York) under the same conditions at a resolution of 2040 1536 pixels, directly coupled to the microscope (BX51, Olympus America, Inc.) and stored in a TIFF file. To quantify the smooth muscle tissue we use the Color Segmentation of ImageJ software, where the program selects structures of different colors and calculates the amount of each component (Figs. 8.7 and 8.8). For quantification of elastic fibers and nerves we use the ImageJ software to determine the volumetric density (Vv) of each component. Results for each field are obtained through the quantification assessment method, by superposing a 100 points test grid (multipurpose test system) on the video monitor screen. The arithmetic mean of the quantification in five fields of each section is determined. Afterward, the mean quantification value for the five sections studied from each sample (total of 25 test areas) is obtained (Fig. 8.8). In order to quantify the area of collagen fibers, elastic fibers, blood vessels, and nerves, a plug-in cell counter and a point tool are used that allow for the quantification of more than one structure in the same photography. The quantity of each analyzed structure is presented in the cell counter window, where the values are tabled and the media obtained for each patient for statistical analysis (Fig. 8.9). For the analysis of the connective tissue and elastic system fibers, photographs of the slices stained by the histochemistry techniques are used: Masson trichrome and Weigert resorcin-fuchsin with previous oxidation, respectively. In both analyses, the microphotographs are obtained under 600×, and five random fields are analyzed by section. For the analysis of blood vessels and nerves, microphotographs of slices stained by the immunohistochemistry method are used: immune-labeling with anti-CD31

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Fig. 8.7  Quantification methods: to quantify the smooth muscle tissue we use the Color Segmentation of ImageJ software, where the program selects structures of different colors and calculates the amount of each component. After calibration and measurement of the image area (red circle 39737.034), select the options plugins, analyze, and grid

Fig. 8.8  Quantification of muscle tissue with color segmentation of ImageJ software – grid window configuration overlapping the microphotograph

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Fig. 8.9  The figure shows the steps to quantify the collagen fibers. Use of the point tool (red circle) and the result of density of collagen fibers is shown in the cell counter window (green rectangle). For quantification of collagen fibers ,the plugin cell counter and the tool point are used that allow for the quantification of more than one structure in the same microphotograph. The quantity of each analyzed structure is shown in the cell counter window, where the values are tabulated and the media obtained for each patient for statistical analysis. Microphotograph, 60×, software ImageJ

and anti-tubulin βIII, respectively. In both analyses, the microphotographs are obtained under 400×, and five random fields are analyzed by section, totalizing 35 fields in control group and 70 fields in the stained group.

8.4  Scanning Electron Microscopy We studied the genital and urinary organs of the fetuses analyzed. For qualitative analysis of connective tissue, we studied five samples from each foreskin, with 2 mm length. The samples are submitted to fixation for scanning electron microscopy (SEM) by immersing tissue fragments in a modified Karnovsky solution for 48 hours at 4 °C. This fixative consists of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. To better visualize the three-­ dimensional organization of the vesicle stroma under SEM, tissue samples are submitted to an alkali treatment to solubilize and remove cells. The obtained

8  Methods of Basic Research Applied to Urinary and Genital Systems… Fig. 8.10 (a) Scan electronic microscopy of fetal renal pelvis. Fetal renal pelvis of a 18-week male fetus postconception. (b) The figure show the ultrastructure of the fetal renal pelvis studied with scan electron microscopy

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acellular preparations are then processed for high-vacuum SEM, and observations are performed on a LEO 435 (Zeiss, Oberkochen, Germany) scanning electron microscope with an acceleration voltage of 15–20 kV (Fig. 8.10).

8.5  Injection/Corrosion Techniques The injection corrosion techniques using resins and anatomic models are very important for translational research. These techniques allow for the tridimensional study of several organs, the study of micro-vascularization, analysis of anatomic relations in humans, and experiment and animal models (Sampaio 2000). Resins are polymers capable of producing solid and saturated compounds (anatomic models). The ideal resin should be cheap, with minimal retraction, producing a strong and consistent mold with unchanged color and easy to manipulate (Tompsett 1970).

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There are several kinds of resins: plastic material, synthetic resins, and silicon resins. The plastic include: nylon, vinylite and Justi h. This kind of resin shows too much retraction (distortion), is fragile, changes its color, and needs a high pressure for injection, complicating its routine use. The synthetic resins include Resapol T208 and Perspex tensol, routinely used in our laboratory with great experience. This class of resin is very resistant to caustic agents. We obtained an easy viscosity regulation with minimal retraction, and they have a low cost. We routinely use Resapol. It is composed by resin, a styrene monomer, a catalyzing agent, and a dye (pigment paste). The styrene monometer allows for the copolymerization and produces a mixture with good viscosity. The catalyzing agent (methyl ethyl ketone peroxide) stiffens the resin, a fundamental step for the confection of molds. The catalyzing agent is liquid, easy to mix, and unstable, with a short limit time for use, and bubbles indicate deterioration. In order to perform the injection, we use the following method: for each 100 ml of resin we add 10 ml of styrene monomer and 2–5 ml of catalyzing agent and the dye (we standardized the following colors: yellow for the collecting system, red for arteries, and blue for veins). Following the hardening of the resin, we initiate the process of corrosion in order to remove all organic material and confection of the mold (Fig. 8.11). After injection, the material must be dipped in hydrochloric, sulfuric, or muriatic acids for 24 hours. After this time, the mold must be removed from the recipient, cleaned, and dried for analysis (Sampaio and Mandarim de Lacerda 1988; Sampaio and Aragao 1992). We have a great experience with this injection technique in the analysis of the intrarenal anatomy with three-dimensional polyester resin endocasts of the kidney collecting system and vessels. We always analyze fresh kidneys from human cadavers, not fixed, with no apparent anomalies of the urinary system. The material is obtained in necropsies performed after 6–24  hours of death. The ureters, renal

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Fig. 8.11  Injection/corrosion techniques: (a) The figure shows a retroperitoneal bloc with the kidneys before the injection; (b) final aspect of renal endocasts after corrosion of organic matter

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arteries, and veins are dissected and injected with yellow, red, and blue resin (Resapol T-208®), respectively, to obtain three-dimensional endocasts. The resin is injected into the ureter, artery, or vein to fill the kidney collecting and vascular systems. A styrene monomer is added to the resin as diluent and methyl ethyl ketone peroxide as catalyst. For each 100 ml of resin, we added 10 ml of styrene monomer, 2.5 ml of catalyst, and 2 ml of yellow pigment. After the injected resin had set, the kidneys are immersed in hydrochloric acid until total corrosion of the organic matter to obtain the endocasts. The endocasts are then analyzed (Figs. 8.12 and 8.13). Following the injection, the kidneys are cleaned and the morphometric measures are obtained, including: length, width of superior pole, width of inferior pole, and a

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Fig. 8.12  The figure shows the steps to prepare fetal kidney endocasts: (a) urogenital organs before dissection, (b) endocast of a fetal kidney arterial system, (c) endocast of fetal kidney vein system

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Fig. 8.13  The figure shows the final aspect of vascular endocasts: (a) endocast of abdominal aorta and renal arteries, (b) final aspect of an endocast of renal arterial tree

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hilar length and width. All measures are obtained by a pachymeter with precision of 0.01 cm. Silicon resin Microfil can also be used, particularly when the purpose is to highlight the organ vasculature. This kind of resin has high cost and is difficult to obtain. We use it in special to study the renal and testicular vasculatures. By thoracostomy, we identify the thoracic aorta and inject the resin inside the vessel (Fig. 8.14). After injection, the abdominal cavity is open and with the aid of a stereoscopic magnifying glass we carefully dissect the organ vessels (Fig. 8.15).

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Fig. 8.14  Silicone (Microfil) resin injection technique: (a) the fetal thoracic cavity is open and the descendent aorta is catheterized; (b) injection of the Microfil resin (with color) in aorta

Fig. 8.15  Final aspect of the fetal organs after the silicone (Microfil) resin injection: (a) Fetus aged 18 weeks postconception: we can observe the testis and the gubernaculum; the vessels are more evident after the resin injection; (b) the renal arteries of a fetus aged 20 weeks postconception after the silicone resin injection

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References Bancroft JD, Cook HC.  Manual of histological techniques and their diagnostic application. Edinburgh: Churchill Livingstone; 1994. Beesley JE. Immunocytochemistry. A practical approach. Oxford: Oxford University Press; 1993. Bradburry P, Rae K. Connective tissues and stains. In: Theory and practice of histological techniques. 4th ed. New York: Churchill Livingstone; 1996. Casey ML, Carr BK. Growth of the kidney in normal human fetus during early gestation. Early Hum Dev. 1982;6:11–4.

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Chagas MA, Babinski MA, Costa WS, et al. Stromal and acinar components of the transition zone in normal and hyperplastic human prostate. BJU Int. 2002;89:699. Cotta-Pereira G, Rodrigo FG, David-Ferreira JF. The elastic system fibers. Adv Exp Med Biol. 1977;79:19–39. Drumm JE, Clinch J, Mackenzie G. The ultrasonic measurement of fetal crown-rump length as a method of assessing gestational age. Br J Obstet Gynaecol. 1976;83:417–21. Favorito LA, Sampaio FJB.  Anatomical relationships between testis and epididymis during the fetal period in humans (10 to 36 weeks postconception). Eur Urol. 1998;33:121–3. Fulmmer HM.  Differential staining of connective tissue fibers in areas of stress. Science. 1958;127:1240–4. Gomori G. Silver impregnation of reticulum in paraffin sections. Am J Pathol. 1937;13:993–1002. Hern WM.  Correlation of fetal age and measurements between 10 and 26 weeks of gestation. Obstet Gynecol. 1984;63:26. Junqueira LCU, Bignolas G, Brentani R. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J. 1979;11:445–7. Mandarim-de-lacerda CA.  Stereological tools in biomedical research. An Acad Bras Cienc. 2003;75:469. Mandarim-de-Lacerda CA, Fernandes-Santos C, Aguila MB. Image analysis and quantitative morphology. Methods Mol Biol. 2010;611:211. Mercer BM, Sklar S, Shariatmadar A, et al. Fetal foot length as a predictor of gestational age. Am J Obstet Gynecol. 1987;156:350. Montes GS, Cotta-Pereira G, Junqueira LCU.  The connective tissue matrix of the vertebrate peripheral nervous system. Adv Cell Neurobiol. 1984;5:177–218. Platt LD, Medearis AL, DeVore GR, et al. Fetal foot length: relationship to menstrual age and fetal measurements in the second trimester. Obstet Gynecol. 1988;71:526. Sampaio FJB. Renal anatomy: endourologic considerations. Urol Clin North Am. 2000;27:585–607. Sampaio FJ, Aragao AH. Inferior pole collecting system anatomy: its probable role in extracorporeal shock wave lithotripsy. J Urol. 1992;147:322–4. Sampaio FJB, Favorito LA. Analysis of testicular migration during the fetal period in humans. J Urol. 1998;159:540. Sampaio FJ, Mandarim de Lacerda CA. Anatomic classification of the kidney collecting system for endourologic procedures. J Endourol. 1988;2:247–51. Spencer RP, Coulombe MJ.  Observations on fetal weight and gestacional age. Growth. 1964;28:243–7. Streeter GL. Weight, sitting height, head size, foot length and menstrual age of the human embryo: Contributions to embryology, vol. 11. Washington, D. C.: Carnegie Institution of Washington; 1920. p. 143–70. Tompsett DH. Anatomical techniques. 2nd ed. Edinburgh: E.& S. Livingstone; 1970. p. 96. True LD. Atlas of diagnostic immunopathology. Philadelphia: JB Lippincott-Co; 1990.

Chapter 9

Basic Research Applied to Hypospadias Luciano Alves Favorito

9.1  Penile Embryology The knowledge of the embryology of the penis allows for a better understanding of several congenital anomalies such as hypospadias, epispadias, and phimosis. At the end of the first month of pregnancy, the posterior intestine and the future urogenital system reach the surface of the embryo at the region of the cloacal membrane ventrally. The cloacal membrane is divided by a septum forming a posterior half (anal) and an anterior half (urogenital membrane) portions. Three bulges arise around the urogenital membrane. The more cephalic is the urogenital tubercle, and the other two (genital bulges) border the urogenital membrane at each side (Fig. 9.1). At this point, male and female genitalia are similar (undifferentiated phase) (Stephens et al. 2002). Under the influence of testosterone that responds to the release of the luteinizing hormone by the hypophysis, the external genitalia is masculinized. One of the first signs of masculinization is the increase of the distance between the anus and the genital structures, followed by the elongation of the penis, formation of the penile urethra from the urethral groove, and development of the prepuce (Fig. 9.2). The development of the prepuce begins at the 13th week of pregnancy, from a circular invagination of the ectoderm that covers all glans at the 20th week of pregnancy (Fig. 9.3). There are three separated portions of the male urethra. The portion above the opening of the Wolff duct (mesonephric) forms the urethra until the verumontanum, including utriculus and urogenital sinus. The second portion forms the segment from the verumontanum to the glans. The glans segment is formed separately. There is an endodermic coating until the bulbar enlargement of the urogenital sinus. From this point, the urethra is coated by ectoderm (Baskin 2000a, b; Duckett and Baskin 1996; Stephens et al. 2002). L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_9

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Fig. 9.1  Male genitalia origin. (a) Undifferentiated stage; (b) elongation of genital tubercle to form the penis; (c) male genitalia totally developed

The urogenital sinus begins at the opening of the Wolff and Muller ducts (paramesonephric) and extends to the urogenital membrane, separating it from the cloacal fossa above. The elongation of this sinus follows the growth of the urogenital tubercle. Under its ventral aspect, a longitudinal groove arises, corresponding to the beginning of the second portion of the urethra (Baskin 2000a, b; Duckett and Baskin 1996). The endodermic urethral plate invades the mesodermal substance of the primitive penis that is coated by the exterior ectodermal epithelium. The urethral groove presses the urethral plate and the coating ectoderm on the groove regresses, exposing the endoderm of the urethral plate. The ends of the urethral plate are united to the ectoderm edges of the groove. The groove deepens, forming the secondary (definitive) urethral groove that is coated by endoderm and flanked by the urethral folds. The endodermic urethra is located inside the mesoderm that is involved by the

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a

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Fig. 9.2  Formation of penile urethra, glans, and foreskin, ventral view. (a) Open urethral plate, (b) progressive fusion of the folds of the urethral plate, (c) urethral orifice located close to coronal sulcus, (d) and (e) fusion of preputial folds to glans, (f) final stage of formation of foreskin and urethra. (Based on Stephens et al. 2002)

ectoderm. After the fusion of the urethral folds, the mesenchyme inside forms the corpus spongiosum (Wiswell 2000). The ectodermal urethral folds that initiate close to the anus fuse over the urethral plate to form the penile urethra. The distal urethra (in the coronal groove) is the last

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Fig. 9.3 Microphotography of sagittal cross section of distal portion of a fetus with 16 weeks, observing that the foreskin (P) covers almost all glans (G), except the distal portion (arrows)

to close. The local of the fusion of the urethral folds forms the perineal raphe (Fig. 9.1). The urethral gutter closes through the involvement of its borders, while the urogenital membrane of the cloacal fossa closes behind, forming a urethral tube that is open in the bladder and in the exposed urethral gutter. The epithelial invagination of the proximal urethra subsequently forms the prostatic lobes (Baskin 2000a, b; Duckett and Baskin 1996; Stephens et al. 2002). Approximately at the same time, the abdominal wall closes anteriorly and all ventral fusion process completes around the 12th week (first trimester). At this moment, the labia-scrotal folds are evident and their fusion at the midline forms the perineal median raphe, extending from the anus through the median line of the scrotum to the glans (Baskin 2000a, b; Wiswell 2000). The glans segment of the urethra that will be part of the navicular fossa is formed later than the penile urethra and by a different mechanism. A groove in the inferior surface of glans is formed, but only its proximal portion is reached by the endodermic urethral plate. An ectoderm plug at the extremity of glans invades the mesenchyme, such as an ectodermic intrusion. As the ectoderm penetrates the glans, it develops a lumen. At the same time, the urethral folds involve more the urethral plate. The ventral segment of the ectodermic intrusion is located dorsally to the distal end of the advanced urethral plate. The primary urethral folds proliferate and close over the groove. The ectodermic intrusion contacts the end portion of the urethral plate, forming the urethral roof. The new ectodermic lumen continues with the proximal endodermic portion of the urethra. Therefore, the dorsal wall of the navicular fossa is formed by ectoderm and the ventral wall by endoderm, explaining why there is a stratified

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Fig. 9.4 Photomicrography of a cross-sectional view of the penis of a fetus with 24 weeks of pregnancy, showing formed penile structures – corpus spongiosum, urethra, and corpora cavernosa. Masson trichrome ×40

squamous epithelium (from ectoderm) at the more distal part of the urethra (Baskin 2000a, b; Duckett and Baskin 1996; Stephens et al. 2002). The length of the penis increases with the pregnancy age, from approximately 6 mm at the 16th week to 26.6 mm at the 38th week, in studies performed by ultrasound (Perlitz et al. 2011; Sharony et al. 2012). In a recent study with human fetuses without anomalies, it was demonstrated that the penile growth correlates with the increase of the area of the corpora cavernosa and spongiosum between the 13th and 36th weeks of pregnancy and that the rhythm of growth was more intense during the second trimester. This fact corroborates the findings of our control group that showed that the penis increased significantly according to the gestational age at linear regression (Gallo et al. 2013). Hypospadias presents an atrophic urethral depression at the local where the normal meatus would be located, and this is explained by anomalies of the ectodermic intrusion. This is the last stage of the formation of the urethra and it explains why there is a great number of hypospadias where the meatus opens in the sub-coronal region (Baskin 2000a, b; Duckett and Baskin 1996; Stephens et al. 2002). In fetuses with 14 weeks of pregnancy, the urethral folds are still not closed and the vacuolized ectodermic urethral plate will form the glans urethra. After the fusion of the urethral folds, the mesenchyme in the interior forms the corpus spongiosum. In 24-week-old fetuses, the urethra is located in the definitive position, and the peri-­ spongiosum fascia and the albuginea are already formed, and cavernous arteries in the corpus cavernosum are also found (Fig. 9.4).

9.2  Foreskin Embryology The foreskin is a mucocutaneous specialized tissue that covers the glans of the penis; it is richly vascularized and innervated and its function is to protect the glans from irritant effects of the urine and feces (Cold and Taylor 1999). The foreskin has

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five layers: mucus with squamosal epithelium, lamina propria, dartos, dermis, and an external cutaneous layer (Cold and Taylor 1999). Collagen and elastic fibers are the fibrotic components of the cellular matrix and are related to pathological alterations in different tissues, including the foreskin (Ushiki 2002; Favorito et al. 2012). The development of foreskin is observed in the end of the third month of pregnancy and completes in the fifth month, around the 18th week postconception (Maizels 1992; Sadler 1995). The formation of the foreskin is directly related to the formation of the glans and glans urethra (Altemus and Hutchins 1991). Patients with anomalies of the urethra formation show alterations of the structure of foreskin. The most common urethral malformation is hypospadias, affecting 1/125W to 1/250 births (Retik and Borer 2002). At hypospadias, besides the ectopic position of the urethral meatus, the ventral foreskin is not developed and the patients present an excess of dorsal foreskin (Fig. 9.5). The main characteristics of hypospadias include

Fig. 9.5  Foreskin aspects in hypospadias. (a) Distal hypospadias with a short ventral foreskin; (b) glans hypospadias with a characteristic ventral foreskin; (c) mega-meatus with an intact foreskin, a hypospadias variation; and (d) penoscrotal hypospadias with a chordee and ventral foreskin

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ectopia of urethral meatus, curvature in different grades of the body of the penis, and incomplete formation of the foreskin at the ventral region of penis (Retik and Borer 2002; van der Putte 2007) (Fig. 9.5). This association of foreskin alteration and urethral ectopy confirms that both present a simultaneous and dependent growth. If by any reason the urethra does not close, the foreskin does not complete its development, and it does not covers the whole glans, and in those cases the foreskin frenulum is not formed (Maizels 1992). Most hypospadias are distal, and the urethral orifice is located at the sub-coronal region or in the medium region of glans (Retik and Borer 2002) (Fig.  9.5). The events that form the distal part of the urethra occur at the end of the first trimester until the fifth month of pregnancy (Maizels 1992; Altemus and Hutchins 1991). Our sample is composed of fetuses with 13–19 weeks postconception, the more important period for the formation of glans urethra and foreskin. There are reports of patients with hypospadias with normal shape of foreskin, without alteration of development (Snodgrass and Khavari 2006), and there are also reports of foreskin agenesis with normal development of urethra (Temiz and Akcora 2007). These situations are very rare, particularly agenesis of foreskin (apostia) with normal urethra, existing only one case described in the literature (Temiz and Akcora 2007). Usually in hypospadias, the urethral anomaly is associated to incomplete formation of foreskin. In order to analyze the chronology of the formation of foreskin, we studied 12 human fetuses with 13–19 weeks postconception, weighing 70–340 g and vertex-­ coccyx length of 11–18.5 cm. In fetuses with 13–14 weeks, the foreskin does not cover totally the glans penis. With 16 weeks, the foreskin covers almost the whole glans, except its distal end (Fig. 9.3). In fetuses with 18–19 weeks, the foreskin covers all glans. The development of the foreskin and the formation of the frenulum occur along with the development of the glans urethra. The development of the frenulum is shown in Fig. 9.6. At the 16th week, the frenulum is not totally developed (Fig. 9.6a, b). In the fetuses with 18  weeks of pregnancy, the foreskin frenulum finishes its development and attaches to the ventral portion of the glans (Fig. 9.6c). In all studied penises, we have observed the presence of the foreskin lamella, a mesenchymal tissue that actively acts in the development of the foreskin and the glans urethra. The preputial lamella is present in higher quantity in fetuses where the foreskin is still not well developed. In the fetuses with higher age, it is observed a lower quantity of mesenchymal tissue in the preputial space. Figure 9.7 shows the characteristics of the preputial lamella during the second trimester of pregnancy. It is present in great quantity in the third gestational trimester. In the figure, we observe a cross-sectional view of the penis of a fetus of 16 weeks showing a mixture of preputial lamella and urethral plate. In this stage of formation of the glans urethra, the urethral plate is invaded by the mesenchyme of the preputial space that canalizes and unites with the penile urethra. The preputial frenulum is a mesenchymal tissue involved by epithelium that unites the foreskin with the ventral region of the glans (Maizels 1992; Altemus and Hutchins 1991). The frenulum is formed by the union at the midline of the

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Fig. 9.6  In this photography, cross-sectional views of the central and distal regions of fetal glans and foreskin show chronologically the formation of the frenulum. (a) Microphotography of a 16-week fetus, stained with hematoxylin and eosin, 100×. In the central portion of glans, the urethral plate (UP) and glans urethra (*) in formation are observed. The frenulum (Fr) has not completed its development. (b) Microphotography of an 18-week fetus, stained by Van Gieson, 40×. The central portion of glans shows complete formation of frenulum (Fr) and glans urethra (*). (c) Microphotography of an 18-week fetus stained by Van Gieson, 200×, showing the frenulum (Fr) attached to the penile glans. L preputial lamella

frenulum-preputial folds. The region of the preputial frenulum divides the glans urethra in two segments: proximal, derived from the penile urethra, and distal, with a controversial origin. There are two theories to explain the formation of the glans urethra: the theory of ectodermic intrusion (Altemus and Hutchins 1991) and the theory of endodermic differentiation (Kurzrock et  al. 1999), which are more accepted nowadays. In our study, we have observed that the preputial frenulum is not totally formed in fetuses of 16 weeks; however, it is completely formed in fetus of 18 weeks, fixed

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Fig. 9.7  Mesenchymal tissue characteristics (preputial lamella) located between the foreskin and glans (preputial space). Microphotography of the penis of a 16-week fetus stained by Masson trichrome 100×. It is possible to observe the great quantity of mesenchymal tissue (preputial lamella – L), communicating to the region of the urethral plate (UP) still not totally canalized. * Glans urethra

at the ventral region of glans, in a stage where the proximal glans urethra has already finished its development. The preputial lamella is a structure actively involved in the formation of the foreskin, glans, and urethra (Maizels 1992; Altemus and Hutchins 1991). This structure is formed by mesenchyme that fills the space located between the glans and the foreskin (preputial space) and in certain phases of development extends to the region of the urethral plate. The preputial lamella was present in all fetal penis in our sample, and in those with lower age (13–16 weeks) it was present in higher quantity than in fetuses with more age (18 and 19 weeks). Our findings suggest that in fetuses with higher gestational age, in the space between the glans and foreskin (preputial space), there is lower quantity of mesenchymal tissue that would progressively predispose to fusion of foreskin and glans. This lowering of mesenchymal tissue during embryonal development could explain the fact that at birth there is a fusion between the glans and foreskin, preventing, in most cases, the exposure of the foreskin. With time, there is a peeling of this epithelial fusion in most children, allowing the exposure of the glans (Baskin 2000a, b). The chronology of the formation of the foreskin in the second trimester is summarized in Table 9.1. It is a rapid process that lasts around 5 weeks, and it is related to formation of the glans urethra. In the beginning of the second trimester, in a fetus with 13 weeks, the foreskin covers the central part of the glans; at 16 weeks, when the glans urethra completes its formation (Maizels 1992; Baskin 2000a, b), the foreskin extends to the distal portion of glans, and in this phase, the frenulum is not totally developed. In fetuses with 18 and 19 weeks, the frenulum is completed and the foreskin covers the whole glans; in the 13th week of pregnancy, the foreskin

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Table 9.1  Developmental steps of the foreskin, frenulum, preputial lamella, and glandular urethra in fetuses during the 13th to 19th weeks postconception (WPC) Age 13–14 WPC 15–17 WPC 18–19 WPC

Foreskin Covers central portion of glans Covers most of glans Covers all of lamella

Frenulum Has not begun to form Is forming

Lamella Large amount Large amount Formation complete Small amount

Glandular urethra Is forming Is forming Formation complete

covers only the central region and the end of formation of foreskin and frenulum is observed around the 18th gestational week. The mesenchymal tissue that fills the preputial space tends to lower in fetuses with higher gestational age, approaching the glans and foreskin. True phimosis is characterized by the inability to expose the glans. At birth, about 95% of male children have phimosis and 90% of them have the problem solved spontaneously around 3 years old (McGregor et al. 2005). When phimosis is not solved, the lack of treatment can lead to obstruction of urine flow, local infection, and penile cancer (Huntley et al. 2003; Nascimento et al. 2011). In some cases, the alternative to surgery is the treatment with topic corticosteroids with or without hyaluronidase (Orsola et al. 2000; Elmore et al. 2002). The topic treatment of phimosis presents good results with efficacy ranging from 67% to 95% in literature (Chu et al. 1999; Wright 1994). Alterations in the structure of extracellular matrix of the foreskin can occur in patients with phimosis due to the ballooning of the foreskin during urination and local infections (Zampieri et al. 2007). Excess of collagen fibers is one of the structural changes in this case (Zampieri et al. 2007). Collagen provides tensile strength and elastin provides tissue elasticity, which can be helpful for tissue compliance (Kim et al. 1991). The increasing concentrations of elastic fibers are related to a larger widening of the tissue. For an easy exposure of the glans, the prepuce needs a larger concentration of elastic fibers (Cold and Taylor 1999). Elastic system fiber alterations are involved in fibrotic tissue formation.

9.3  Histologic Study of the Foreskin in Hypospadias Hypospadias repair is a frequent surgery for the pediatric urologist. The incidence of complications such as fistulas, urethral and meatal stenosis, and scar formation are not uncommon (Davits et al. 1993; Luo et al. 2003; Roth et al. 2008). Besides the clinical aspect, it is also important to evaluate the psychosexual aspect, which is compromised in a great number of the patients, especially due to the cosmetic aspects of the penis (Mureau et al. 1995; Scarpa et al. 2009); also, the foreskin is very important to the surgery success. Although androgen stimulation with testosterone, dihydrotestosterone, or human chorionic gonadotropin has been applied in order to improve the aesthetic outcome

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and reduce complications as they temporarily promote penile growth, facilitating surgical correction (Davits et al. 1993; Kaya et al. 2008; Bastos et al. 2011), its real effects on the prepuce to improve postoperative results are still not fully understood. Studies on foreskin structure in patients with hypospadias are scarce. Recently, we demonstrated that the use of topical 1% testosterone propionate before hypospadias surgery produces foreskin neovascularization in absolute numbers and in volume density (Cold and Taylor 1999). Also, to our knowledge, there are no studies on histological alterations of the foreskin collagen and elastic fibers in patients with hypospadias after treatment with topical testosterone. The present study aims to evaluate the effect of topical 1% testosterone propionate on elastic and collagen fibers, which have important participation in the healing process. A previous study in a prospective randomized study in prepubescent children with hypospadias studied the use of testosterone and the prepuce histological alterations in patients with hypospadias (Paiva et al. 2016). The children were divided into three groups: control (group 1); hypospadias without testosterone (group 2), and hypospadias with testosterone (group 3). The groups 2 and 3 were divided at random using the program Research Randomizer (www.randomizer.org). This study concluded that children treated with testosterone showed a less homogeneous pattern and lower surface density of collagen fibers than that observed in untreated children. There was no difference between groups regarding surface density of elastic fibers, but there was a trend to increase in the group treated with testosterone. Elastic fibers were more homogeneous in patients who received testosterone. The foreskin of children with hypospadias has worse quality of elastic fibers than normal foreskin. Collagen and elastic fibers are the fibrotic components of the cellular matrix and are related to pathological alterations in different tissues. Collagen and elastic fibers are important components of the prepuce. Collagen provides tensile strength and elastin provides tissue elasticity, which can be helpful for tissue compliance. The increasing concentrations of elastic fibers are related to a larger widening of the tissue. For an easy exposure of the glans, the prepuce needs a larger concentration of elastic fibers (Favorito et al. 2012). Elastic system fiber alterations are involved in fibrotic tissue formation. In a previous study, we observed significant histological alterations in the prepuce in patients with phimosis submitted to topical treatment, with corticosteroids + hyaluronidase (Franck-Lissbrant et al. 1998). Patients in whom topical steroid treatment failed had fewer elastic fibers, which is a characteristic of the healing processes, and an amplification of the collagen type III, a recently found collagen that is associated with muscular retraction (Franck-Lissbrant et al. 1998).

9.4  Penile Structure in Human Fetuses The urogenital cavity extends to the surface of the urogenital fold, forming a linear endodermic membrane, around the sixth week of pregnancy. This membrane is temporarily filled with an endodermic structure called urethral plate that disintegrates temporarily to then become more adherent to the structures of the future genitalia. In male embryos, this membrane is more elongated and broader, while in female

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embryos present a shorter structure and more angulated behavior. Both embryos present an ectodermic bud in the end of the urethral plate. The genital tubercle elongates to form the phallus, and primordial clitoral and penile glans are delimited in the phallic shaft through the coronal sulcus. The external genitalia appearance is similar in both embryo sexes until the seventh week of pregnancy (Maizels 1992). In the fourth month of pregnancy, the effects of dihydrotestosterone in male embryos manifest by an increase of the separation of the urogenital sinus from the anal-rectal canal. The labia-scrotal folds fuse medially to form the scrotum, and the urethral folds fuse to originate the penile urethra that is completely formed in the 14th week of pregnancy. The distal penile urethra is formed by the combination of the fusion of the urethral plates and the interiorization of the ectodermic cells located in the distal segment of the penile glans. In the adult, this embryologic difference is manifest by the difference of epithelium found in the penile urethra (median and posterior thirds) in a pseudostratified pattern, while in glans urethra a squamous coating epithelium is observed. It is speculated that an intense mechanism of interactivity between the urethral mesenchyme, hormones, and neurotransmitters is necessary, mediated by the action of dihydrotestosterone (Maizels 1992). In the absence of dihydrotestosterone, the primitive perineum does not elongate and the labia-scrotal folds don’t fuse in the midline. The phallus moves inferiorly forming the clitoris, and the definitive urogenital sinus transforms in the vaginal vestibule. The urethral folds will form the minor labia and the labia-scrotal leaflets will form the major labia. Male fetuses without 5-alpha-reductase or those with alterations of metabolism of testosterone or dihydrotestosterone may present a phenotypic similar development. The role of the gene HOX4 on the phenotypic differentiation of the urogenital system in males is also known (Ravasi et al. 2010). The urethral development happens in parallel to the development of the penile shaft. Anatomically, the urethra is divided into two major segments: anterior and posterior. The anterior segment has approximately 15 cm length and extends from the urethral meatus to the bulbar urethra. The posterior urethra is formed by the membranous and prostatic segments. It is intimately related to all organs of the male genital system, sharing reproductive and urinary excretion proprieties. The embryologic development of the most posterior segment of male urethra is similar to the bladder trigone and prostatic urethra. The development of the anterior segment of the urethra is intimately related to the development of the phallus. Since the urethral meatus does not present tissue with specific embryological origin, the initial development of the pendular urethra may present two urethral primordia called urethral plates that join in a blind point. The analysis of the penile and urethra structures in the fetus was made by histochemistry and immunohistochemistry of human fetuses of the second trimester of pregnancy (Fig. 9.8). Early studies of normal human fetuses proved that the collagen concentration increased progressively and almost doubled from the 17th to 33rd weeks, suggesting that major changes of the extracellular matrix of the penile shaft occur during

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Fig. 9.8  Photography of the penis of a second trimester fetus used for the histology study of the penile structure

Fig. 9.9 Microphotography showing connective tissue and smooth muscle cells in the penis of a normal fetus with 20 WPC. The corpora cavernosa (Cc) and the tunica albuginea (Ta) can be seen. Masson’s trichrome ×200

that gestational period. These changes could be related to the erectile function (Bastos et al. 2004). These results reinforce the fact that, with fetal genitalia maturation, there is a gradual increase of the collagen concentration, arranging the penile shaft elements for erection. The concentration of soft muscular cells decreased with the advance of fetal maturity. Using immunohistochemistry and comparing the distribution of soft muscular cells, it was possible to observe their prevalence in the lumen of the sinusoids of the corpus cavernosum (Figs. 9.9 and 9.10). In the corpus spongiosum, the muscular fibers are located more eccentrically in relation to the urethral lumen. This divergent distribution could be explained by the physiology of each structure: the

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Fig. 9.10 Microphotography showing immunohisto­ chemistry of the smooth muscle cells (arrow) in the cavernosa trabeculae in a normal fetus with 18 WPC. Myosin immunolabeling ×400

Fig. 9.11 Microphotography showing spongiosum tissue and urethral lumen of the fetal penises in a fetus with 16 WPC. The urethral lumen and the well-­preserved urethral epithelium can be seen. Masson’s trichrome ×200

corpus cavernosum needs more distensibility in order to maintain the blood in the sinusoids; in the corpus spongiosum, the eccentric distribution of the muscular fibers would be related to the eventual peristaltic movement produced by the urethra. Also, the lowering of concentration of muscular fibers also expresses as an increase of thickness of tunica albuginea, rich in collagen, with the progress of gestation age. When the corpus cavernosa and spongiosum were evaluated, there were no changes of collagen density and soft muscle in relation to the increase of gestational age. The urethral epithelium was integrated in the studied samples. Elastin staining was positive only after the 22nd week (Fig. 9.11). We also found that the size and thickness of the elastic fibers in the urethra increased with age, mainly in the third trimester of gestation. The concentration of elastic fibers in the spongy urethra increases significantly with age. The high concentration of elastic fibers in the

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Fig. 9.12 Microphotography showing the elastic system fibers. Immunohistochemistry showing elastic fibers (brown) in the corpus spongiosum of a normal fetus with 20 WPC. Elastin immunolabeling ×400

spongy urethra may partially explain its high extensibility. The progressive increase of elastic fiber concentration during development implies functional adaptation of the fetal male urethra. Preservation of fetal genitalia by exposure of central nervous system to amniotic liquid suggests that the mechanisms of maturation of male genitalia are only dependent of the hormone and molecular interaction. Interaction of androgen and estrogen receptors in target organs regardless of the interaction of central nervous system is well known in study models with rats (Goya et  al. 2009); however studies in humans are rare in literature. The development of elastic fibers may have an important participation in the maturation mechanism of fetal genitalia. Presence of elastin confirmed by immunohistochemistry after the 22nd week suggests that, with the lowering of muscular fibers due to increase of gestational age, the elastic fibers would have a greater participation in the tissue pool (Fig. 9.12). The presence of elastin in fetuses after the 20th week is an objective data of maintenance of erection in these groups. The increase of connective tissue and elastic fibers and decrease of muscular fibers in corpora cavernosa suggest that there is a gradual tissue substitution with fetal maturation.

References Altemus AR, Hutchins GM. Development of the human anterior urethra. J Urol. 1991;146:1085–93. Baskin LS. Hypospadias and urethral development. J Urol. 2000a;163:951–6. Baskin LS.  Hipospadias. Anatomy embriology and reconstructive techniques. Braz J Urol. 2000b;26:621–9. Bastos AL, Silva EA, Costa WS, Sampaio FJB.  The concentration of elastic fibers in the male urethra during human fetal development. BJU Int. 2004;94:620–3.

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Bastos AN, Oliveira LRS, Ferrarez CEPF, et  al. Structural study of prepuce in hypospadays  – does topical treatment with testosterone produce alterations in prepuce vascularization? J Urol. 2011;185:2474–8. Chu CC, Chen KC, Diau GY. Topical steroid treatment of phimosis in boys. J Urol. 1999;162:861–3. Cold CJ, Taylor JR. The prepuce. BJU Int. 1999;83(suppl 1):34–44. Davits RJ, Van Den Aker ES, Scholtmeijer RJ, et al. Effect of parenteral testosterone therapy on penile development in boys with hypospadias. Br J Urol. 1993;71:593–5. Duckett JW, Baskin LS. Hypospadias. In: Adults and pediatric urology. St. Louis: Mosby; 1996. p. 2549. Elmore JM, Baker LA, Snodgrass WT. Topical steroid therapy as an alternative to circumcision for phimosis in boys younger than 3 years. J Urol. 2002;168:1746–7. Favorito LA, Balassiano CM, Rosado JP, Cardoso LE, Costa WS, Sampaio FJ. Structural analysis of the phimotic prepuce in patients with failed topical treatment compared with untreated phimosis. Int Braz J Urol. 2012;38:802–8. Franck-Lissbrant I, Häggström S, Damber JE, et al. Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats. Endocrinology. 1998;1392:451–6. Gallo CB, Costa WS, Furriel A, Bastos AL, Sampaio FJ.  Development of the penis during the human fetal period (13 to 36 weeks after conception). J Urol. 2013;190(5):1876–83. Goya HO, Braden TD, Willians CS, Willians JW. Estrogen-induced developmental disorders of the rat penis involve both estrogen receptor (ESR)- and androgen receptor (AR)-mediated pathways. Biol Reprod. 2009;81:507–16. Huntley JS, Bourne MC, Munro FD, Wilson-Storey D. Troubles with the foreskin: one hundred consecutive referrals to paediatric surgeons. J R Soc Med. 2003;96:449–51. Kaya C, Bektic J, Radmayr C, et  al. The efficacy of dihydrotestosterone transdermal gel before primary hypospadays surgery: a prospective, controlled, randomized study. J Urol. 2008;179:684–8. Kim KM, Kogan BA, Massad CA, Huang YC. Collagen and elastin in the normal fetal bladder. J Urol. 1991;146:524–7. Kurzrock EA, Baskin LS, Cunha GR. Ontogeny of the male urethra: theory of endodermal differentiation. Differentiation. 1999;64:115–22. Luo CC, Lin JN, Chiu CH, et  al. Use of parenteral testosterone prior to hypospadias surgery. Pediatr Surg Int. 2003;19:1–2. Maizels M. Normal development of the urinary tract. In: Campbell’s urology. 6th ed. New York: Saunders; 1992. p. 1301. McGregor TB, Pike JG, Leonard MP.  Phimosis—a diagnostic dilemma? Can J Urol. 2005;12:2598–602. Mureau MA, Slijper FM, van der Meulen JC, et al. Psychosexual adjustment of men who underwent hypospadias repair: a norm-related study. J Urol. 1995;154:1351–5. Nascimento FJ, Pereira RF, Silva JL II, Tavares A, Pompeo ACL. Topical betamethasone and hyaluronidase in the treatment of phimosis in boys: a double-blind, randomized, placebo- controlled trial. Int Braz J Urol. 2011;37:314–9. Orsola A, Caffaratti J, Garat JM. Conservative treatment of phimosis in children using a topical steroid. Urology. 2000;56:307–10. Paiva KC, Bastos AN, Miana LP, Barros Ede S, Ramos PS, Miranda LM, Faria NM, Avarese de Figueiredo A, de Bessa J Jr, Netto JM. Biometry of the hypospadic penis after hormone therapy (testosterone and estrogen): a randomized, double-blind controlled trial. J Pediatr Urol. 2016;12(4):200.e1–6. Perlitz Y, Keselman L, Haddad S, Mukary M, Izhaki I, Ben-Ami M. Prenatal sonographic evaluation of the penile length. Prenat Diagn. 2011;31(13):1283–5. Ravasi T, Suzuki H, Cannistraci CV, Katayama S, Bajic VB, et al. An atlas of combinatorial transcriptional regulation in mouse and man. Cell. 2010;140(5):744–52. Retik AB, Borer JG. Hypospadias. In: Campbell’s urology. 8th ed. New York: Saunders; 2002. p. 2284–333.

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Chapter 10

Basic Research Applied to Renal Anomalies Luciano Alves Favorito, Andre L. Diniz, and Francisco Jose B. Sampaio

10.1  Introduction The second gestational trimester is very important for the embryonic development of the kidneys, renal pelvis, bladder, and ureter (Short and Smyth 2016; Moore and Persaud 2003). An important branching of the ureteric bud occurs between the 5th and 14th weeks postconception (WPC), leading to formation of the major and minor renal calyces, renal pelvis, and collecting tubules (Stephens et al. 2002; Ishiyama et al. 2018). The process of kidney development is completed by the 34th week of gestation in humans (Blake and Rosenblumb 2014). Nephrons start this development in the eighth week and all of the branches of the ureteric bud and the nephron units are formed by 32–36 weeks of gestation; however, these structures are not yet mature and will continue to mature after birth (Short and Smyth 2016). The development of ultrasonography in the 1970s enabled the prenatal diagnosis of fetal malformations during regular monitoring of pregnant women. Birth defects are the leading causes of infant mortality, and in 2013 these were associated with 4778 deaths (20% of deaths in the first year of life) in the United States (Mathews and MacDorman 2013). In a recent 5-year cohort study, researchers reported a L. A. Favorito (*) Urogenital Research Unit - Department of Anatomy, Rio de Janeiro State University, Rio de Janeiro, Rio de Janeiro, Brazil A. L. Diniz Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil F. J. B. Sampaio Urogenital Research Unit from Rio de Janeiro State University, Rio de Janeiro, Brazil National Council for Scientific and Technological Development (CNPq – Brazil), Brasília, Brazil Rio de Janeiro State Research Foundation (FAPERJ), Rio de Janeiro, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 L. A. Favorito (ed.), Translational Research in Pediatric Urology, https://doi.org/10.1007/978-3-030-50220-1_10

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prevalence of 2% of major birth defects with estimate of known etiology in just over 20% of these cases (Feldkamp et al. 2017). Although congenital heart diseases are the most common birth defects and the leading causes of infant death worldwide (van der Linde et al. 2011), renal anomalies have gained space in scientific production and news reports around the world. In this chapter we show important studies about the kidney and renal pelvis applied to renal anomalies. The fetuses studied in this chapter were macroscopically well preserved and gestational age was determined in WPC according to the foot-length criterion. The fetuses were also evaluated regarding total length (TL), crown-rump length (CRL), and body weight. After the measurements, the fetuses were carefully dissected with the aid of a stereoscopic lens with 16/25× magnification. The kidneys were removed together with the ureters, bladder, and genital organs. After kidney dissection, we evaluated the renal pelvis and kidney measurements.

10.2  Kidney Surface in Human Fetuses The surface of the fetal kidney is divided by a number of clefts into lobes and lobules (Fig. 10.1). Fetal kidney lobes (clefts) are fine, linear demarcations indenting the renal surface, separating the normal lobes, consisting of a central pyramid and surrounding cortex. The interlobular boundary lines are apparent as grooves on the surface of the fetal kidney, but are rarely visible in the mature kidney. Studies of renal clefts in human fetuses are rare. We studied 140 kidneys obtained from 70 human fetuses (38 males and 32 females) ranging in age from 12 to 25 weeks post-conception (WPC) (Fig. 10.2). The kidney was dissected and the number of clefts was counted. The renal length was also assessed. To compare the quantitative data in both sexes, the Student’s t-test was used (p