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This article will review the embryological development of the testicles. However, to understand the embryology of the testicles, it is essential to have a basic understanding of the normal anatomy of the testicles. The male gonads, otherwise known as the testicles, are sex glands that have both an exocrine secretory function in the production of sperm and an endocrinological function as part of the hypothalamic-pituitary-gonadal axis in men through the production of androgens. The normal anatomy of the testicles is that of an oval shape located in the scrotum, further separated by the scrotal septum. The length of the testis is between 3 cm to 5 cm, whereas the width is between 2 cm to 3 cm. The consistency of normal testicles on palpation is smooth and soft. The testes are suspended superiorly by the spermatic cord and inferior to the scrotum by the scrotal ligament. During embryological development, the scrotal ligament is also known as the gubernaculum.[1][2] The tunica vaginalis is a double-layered structure that covers all of the testes apart from the posterior and superior borders, which represent the attachment of the epididymis and spermatic cord.  The posterior lateral testis has a small space between the body of the epididymis and the testis. This small space is known as the sinus of the epididymis. The tunica albuginea is found deep in the tunica vaginalis. It is a thick fibrous sheath that covers the testes. The descent of the testicles is a complex stepwise process that involves an interaction between many anatomical structures, environmental influences, regulatory hormones, and inherited genetic factors.

Testicular Developmental Disorders Testicular Dysgenesis Testicular dysgenesis syndrome (TDS) is a constellation of poor-quality semen, testicular germ cell cancer, cryptorchidism, and hypospadias. It is a consequence of disruption to embryonal programming and gonad development.[78] The belief is that testicular dysgenesis syndrome is the result of environmental disrupting chemicals (EDCs), intrauterine growth disorders, genetic and lifestyle factors. Reduced production of androgen receptors, disrupted hormone levels along with exposure to endocrine disrupters are associated with the development of TDS.[79] The thought is that this occurs due to Leydig-cell dysfunction and reduced INSL3 secretion from Leydig cells, which consequently leads to the disorders associated with TDS.[80] Furthermore, a disruption in Sertoli cell development contributes to the development of TDS.[81] As mentioned before, researchers believe that EDCs contribute to the development of TDS through the interference with the production, transport, metabolism, binding, release, action, and elimination of hormones associated with developmental processes.[82] This result can be through anti-androgenic effects, estrogenic effects of steroidogenic enzyme inhibitory effects.[83][84] There are historical examples of maternal exposure to chemicals contributing the testicular developmental disorders; the synthetic estrogenic drug known as diethylstilboestrol (DES) was historically used for the prevention of the complications of pregnancy but was shown to increase the risk of testicular malformations.[85]  Another environmental toxin that appears to contribute to testicular malformations is bisphenol A (BPA). This chemical has weak estrogenic effects and is used in the production of polycarbonates and epoxy resins.[86] Phthalate esters are used in vinyl plastics to improve their flexibility. However, it seems that they have an endocrine-disrupting effect.[79] Cryptorchidism

There are historical examples of maternal exposure to chemicals contributing the testicular developmental disorders; the synthetic estrogenic drug known as diethylstilboestrol (DES) was historically used for the prevention of the complications of pregnancy but was shown to increase the risk of testicular malformations.[85]  Another environmental toxin that appears to contribute to testicular malformations is bisphenol A (BPA). This chemical has weak estrogenic effects and is used in the production of polycarbonates and epoxy resins.[86] Phthalate esters are used in vinyl plastics to improve their flexibility. However, it seems that they have an endocrine-disrupting effect.[79] Cryptorchidism It is a state wherein there is an absence of the testes in the scrotum may be unilaterally or bilaterally. The etiology of cryptorchidism can divide into anatomical, genetic, or hormonal causes.[87][88] Anatomical causes of cryptorchidism include conditions that cause an anatomical abnormality of the inguinal canal, vas deferens, or testis themselves.[89] Genetic causes of cryptorchidism include 5α-reductase gene mutations, androgen receptor mutations, mutations in INSL3 genes.[90][91][90][92] Hormonal causes of cryptorchidism include a deficiency or insensitivity to AMH, INSL3, androgen, or GNRH/LH.[87][93][94] Cryptorchidism is problematic as it can lead to an increased likelihood of infertility and an increased likelihood of developing testicular cancer.[95][93] As the main functional significance of the descent of the testes according to the ‘temperature-dependent hypothesis’ is the maintenance of a low-temperature environment to produce viable sperm, failure of testicular descent will lead to an increase in the testicular temperature. An increase in testicular temperature causes a reduction in effective spermatogenesis. Furthermore, damage to Sertoli cells may occur due to the production of reactive oxygen species.[69][96] Cryptorchidism appears to demonstrate associations with an increased risk of testicular intraepithelial neoplasm (TIN), which may lead to testicular carcinoma in situ (CIS).[97] It is thought that the high temperatures associated with cryptorchidism lead to a failure of the development of neonatal gonocytes to differentiate into primary spermatogonia, which allows some gonocytes to persist as a potential source of malignancy.[95][93] Anorchia

Cryptorchidism appears to demonstrate associations with an increased risk of testicular intraepithelial neoplasm (TIN), which may lead to testicular carcinoma in situ (CIS).[97] It is thought that the high temperatures associated with cryptorchidism lead to a failure of the development of neonatal gonocytes to differentiate into primary spermatogonia, which allows some gonocytes to persist as a potential source of malignancy.[95][93] Anorchia By definition, anorchia is the absence of testes in a person who has a 46 XY karyotype and an external male phenotype. It is also known as vanishing testis syndrome or embryonic testicular regression.[98][99][100] The exact etiology of congenital anorchia is not well known. However, there appears to be a genetic component due to the familial inheritance associated with anorchia.[98] However, in previous studies, mutations in genes such as SRY, INSL3/INSL3 receptor do not have an association with anorchia.[101] But mutations in genes such as NR5A1, which codes for SF1, correlate with anorchia.[102] Because patients with anorchia have a normal male phenotype, scientists suspect that the testes were functionally normal during some point of embryological development.[103] Other suspected etiologies are testicular torsion, vascular occlusion, and trauma during the descent of the testes.[104] Monorchism Monorchism is, by definition, the state of only having one testis in the scrotum. Monorchism may result from trauma, testicular torsion, or surgical removal because of testicular malignancy.[105] Congenital monorchism is when a child is born with only one testis in the scrotum; this can be due to cryptorchidism or vascular pathology, which causes unilateral regression of the testis (anorchia/vanishing testis syndrome) such as spermatic cord torsion or thrombosis.[106][107] Monorchism may also occur in disorders of sexual development (DSD).[108] However, endocrine abnormalities are unlikely to be the sole cause of unilateral congenital monorchism. Men with congenital monorchism do not have a significant difference in hormonal excretory function compared to men with two testes.[109] Polyorchidism

Monorchism is, by definition, the state of only having one testis in the scrotum. Monorchism may result from trauma, testicular torsion, or surgical removal because of testicular malignancy.[105] Congenital monorchism is when a child is born with only one testis in the scrotum; this can be due to cryptorchidism or vascular pathology, which causes unilateral regression of the testis (anorchia/vanishing testis syndrome) such as spermatic cord torsion or thrombosis.[106][107] Monorchism may also occur in disorders of sexual development (DSD).[108] However, endocrine abnormalities are unlikely to be the sole cause of unilateral congenital monorchism. Men with congenital monorchism do not have a significant difference in hormonal excretory function compared to men with two testes.[109] Polyorchidism Polyorchidism refers to a congenital anomaly in which more than two testes are present in the scrotum.[110] Even though this is a very rare condition; 40 % of polyorchidism is associated with undescended testes.[111] The most common type of polyorchidism is triorchidism, which refers to the presence of three testicles.[112] It appears that polyorchidism occurs due to abnormal separation of the genital ridge during the development of the testes in the embryo.[113] Leung described a way of classifying polyorchidism based on embryological differentiation.[114] Type I: An extra testis without an epididymis or vas which attaches to the ipsilateral testis. Type II: The extra testis drains through the epididymis of the ipsilateral testis, and they share a common vas deferens. Type III: The extra testis has its own epididymis separate from the ipsilateral testis, both of those structures draining into a common vas deferens. Type IV: The extra testis has a separate epididymis and vas deferens from the ipsilateral testis. Testicular Torsion Testicular torsion is a state in which the spermatic cord twisted upon itself, leading to a restriction of blood supply and venous drainage of the ipsilateral testis. If emergency treatment is not delivered quickly, it may lead to necrosis of the testis.

Type IV: The extra testis has a separate epididymis and vas deferens from the ipsilateral testis. Testicular Torsion Testicular torsion is a state in which the spermatic cord twisted upon itself, leading to a restriction of blood supply and venous drainage of the ipsilateral testis. If emergency treatment is not delivered quickly, it may lead to necrosis of the testis. Researchers believe that the majority of postnatal testicular torsion is associated with the "bell clapper anomaly."[115] Hypermobility of the testicle is thought to be inherited through an X-linked dominant or Y-linked mechanism.[116][117][118] The bell clapper anomaly refers to the posterior attachment of the parietal lamina of the tunica vaginalis (TVPL). In a normal testis, the TVPL is attached to the posterolateral aspect of the testis, whereas in patients with the bell clapper anomaly, the TVPL attachment encircles and connects proximally to the spermatic cord, which leads to free-hanging testis in the tunica vaginalis.[119] Testicular torsion is extravaginal or intravaginal.[120] Extravaginal testicular torsion occurs more frequently in neonates, it is due to the testis, epididymis, and tunica vaginalis twisting along with the spermatic cord.[121] In contrast, intravaginal testicular torsion occurs secondary to the bell clapper deformity.[120] Swyer Syndrome Swyer syndrome, also known as pure gonadal dysgenesis, occurs when there is abnormal sexual differentiation during embryological development, leading to insufficient intrauterine virilization with undifferentiated gonads with associated female phenotype.[122] The external genitalia is non-ambiguous and well-formed; however, the gonads are dysgenic and improperly formed.[123] Patients with Swyer syndrome have an XY 46 karyotype. Swyer syndrome occurs due to reduced production of testosterone and AMH leading to a failure of gonadal differentiation.[122] Particular mutations associated with Swyer syndrome include mutations in NR5A1 (which encodes for SF1), WNT4, and the SRY gene, which mutates in 10 to 15% of patients with Swyer syndrome.[124][123] Klinefelters Syndrome

Swyer syndrome occurs due to reduced production of testosterone and AMH leading to a failure of gonadal differentiation.[122] Particular mutations associated with Swyer syndrome include mutations in NR5A1 (which encodes for SF1), WNT4, and the SRY gene, which mutates in 10 to 15% of patients with Swyer syndrome.[124][123] Klinefelters Syndrome Klinefelters syndrome is a common congenital chromosomal disorder with an incidence of 0.1% to 0.2% of newborns.[125] It is a common cause of primary hypogonadism.[126] The majority of patients (80%) have the 47 XXY karyotype, whereas 20% of patients with Klinefelter have supernumerary X chromosomes (49 XXXXY or 48 XXXY), mosaicism of 47 XXY/46XY, or more than one Y chromosome (XXYY).[126][127][128] Androgen Insensitivity Syndrome A child can have a disorder of sexual development (DSD) without a chromosomal abnormality such as Klinefelters syndrome. The most commonly seen 46 XY DSD is androgen insensitivity syndrome (AIS).[129] The severity of AIS can range from complete androgen insensitivity syndrome (CAIS), in which female genitalia is present, to mild androgen insensitivity syndrome (MAIS) in which normal external male genitalia is present with or without gynecomastia.[130][131] AIS occurs due to molecular defects in the gene that codes for the androgen receptor (AR).[132] The gene for the AR is on the X chromosome at position Xq11-12.[133] Mutations in the gene that provides information for the AR known as NR3C4 cause AIS.[134] The AR is a nuclear receptor that binds to androgens such as testosterone and dihydrotestosterone (DHT). As patients with AIS have a Y chromosome, they will produce male gonads. However, as the fetal cells do not respond to androgens, there will be abnormal development of the reproductive system. This situation may manifest as undescended testes. Furthermore, as androgens play a part in the inhibition of female characteristics through SOX9 and SF1, a loss of response to androgens may lead to a female phenotype.[135] 5-ARD Deficiency The enzyme 5-alpha reductase 2 (5alpha-RD2) converts testosterone into DHT.[136] As mentioned earlier, androgens are essential during the development of male genitalia. A failure of the conversion of testosterone into DHT will lead to abnormal development of the genitalia.

As patients with AIS have a Y chromosome, they will produce male gonads. However, as the fetal cells do not respond to androgens, there will be abnormal development of the reproductive system. This situation may manifest as undescended testes. Furthermore, as androgens play a part in the inhibition of female characteristics through SOX9 and SF1, a loss of response to androgens may lead to a female phenotype.[135] 5-ARD Deficiency The enzyme 5-alpha reductase 2 (5alpha-RD2) converts testosterone into DHT.[136] As mentioned earlier, androgens are essential during the development of male genitalia. A failure of the conversion of testosterone into DHT will lead to abnormal development of the genitalia. 5-alpha reductase deficiency is a 46 XY DSD. It presents with a female phenotype or improperly virilized external genitalia along with testes. The gene for 5 alpha-RD2, also known as SRD5A2, is found on the second chromosome on position 2p23, it is an autosomal recessive condition.[137][136] Congenital Adrenal Hyperplasia The most common inborn error of adrenal function is congenital adrenal hyperplasia (CAH) secondary to a deficiency of the steroid 21-hydroxylase enzyme.[138] This condition occurs due to deletions or mutations in the gene CYP21A2, which codes for the steroid 21-hydroxylase enzyme found on chromosome 6p21.[139][140] This mutation leads to an impairment of the production of cortisol and aldosterone alongside excessive adrenal androgen production. However, this form of congenital adrenal hyperplasia does not cause anatomical disorders in males. In infant females, the most common form of congenital adrenal hyperplasia (21-hydroxylase deficiency) may present with a salt-wasting disease or virilization of the external genitalia. In both males and females, it can present with precocious puberty.[140] Other less common forms of congenital adrenal hyperplasia include the 3b-hydroxysteroid dehydrogenase (3b-HSD) deficiency which occurs due to mutations in the HSD3B2 gene, the 17a-hydroxylase (P450 17a) deficiency which occurs due to mutations in the CYP17A1 gene, and the P450 side-chain cleavage enzyme (P450scc) deficiency which occurs due to mutations in the CYP11A1 gene.[140]

Other less common forms of congenital adrenal hyperplasia include the 3b-hydroxysteroid dehydrogenase (3b-HSD) deficiency which occurs due to mutations in the HSD3B2 gene, the 17a-hydroxylase (P450 17a) deficiency which occurs due to mutations in the CYP17A1 gene, and the P450 side-chain cleavage enzyme (P450scc) deficiency which occurs due to mutations in the CYP11A1 gene.[140] A deficiency in the 3b-HSD enzyme causes a failure of the conversion of 17-a hydroxypregnenolone to 17a hydroxyprogesterone, pregnenolone to progesterone, and DHEA to androstenedione, resulting in the accumulation of pregnenolone, DHEA, and 17-a hydroxypregnenolone and a reduction in the production of sex steroids. It may also cause salt-wasting disease.[141] Reduced production of androgenic steroid hormones may present as pseudohermaphroditism associated with cryptorchidism.[141] A deficiency in the P450 17a enzyme leads to a failure of the conversion of pregnenolone to 17a-hydroxypregnenolone, progesterone to 17a- hydroxyprogesterone, and reduced conversion of 17-hydroxypregnenolone into DHEA. This process leads to the accumulation of pregnenolone, progesterone, and reduced production of glucocorticoids and sex steroids, leading to incomplete virilization in males associated with ambiguous external genitalia and cryptorchidism.[142][143] A deficiency in the P450scc enzyme leads to a failure of the conversion of cholesterol into pregnenolone; this is the first step in the synthesis of all steroid hormones. As a result, there is a severe deficiency in steroid hormones.[144] Regardless of the chromosomal gender, children with a mutation in P450scc are born phenotypically female.[145] However, due to a failure of the production of androgens, the testes fail to descend.[146] Other Related Conditions Congenital Inguinal Hernia The lifetime risk of inguinal hernias is significantly higher in men (27%) than in women (3%).[147] As such, one of the main risk factors for a primary inguinal hernia is the male gender.[148] Other risk factors for primary inguinal hernias include patency of the processus vaginalis. Obliteration of the processus vaginalis occurs typically by the third trimester through programmed cell death of smooth muscle cells.[149] Non-obliteration of this process vaginalis results in abnormal protruding of abdominal contents through the inguinal canal into the scrotum.