The Normal Function of the Testes

There are two main functions of the testes. These are the endocrine production of androgens and exocrine production of spermatozoa.

The endocrine function of the testis is under neuroendocrine control. The production of gonadotrophin releasing-hormone (GnRH) by the anterior hypothalamus stimulates the production of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the anterior pituitary.[55] LH and FSH bind to receptors found on Sertoli and Leydig cells. Luteinizing hormone stimulates Leydig cells to produce testosterone, whereas FSH stimulates Sertoli cells to produce androgen binding proteins (ABPs) along with the regulation of spermatogenesis.[56]

Endocrine Function

GnRH is released in repeated pulses in response to excitatory neurotransmitters such as glutamate and kisspeptin.[57][58] GnRH binds to a G-protein seven-transmembrane receptor (GPCR) known as the gonadotrophin-releasing hormone receptor (GnRHR) on gonadotrophic cells of the anterior pituitary. FSH becomes stimulated when GnRH pulses are a low frequency, whereas stimulation of LH occurs when GnRH pulses are at a high frequency.[59][60]

Luteinizing hormone stimulates the production of androgens in Leydig cells by binding to a GPCR.[61] During testicular development, blood testosterone levels peak three times. The first time is between 12 to 14 weeks of gestation when Leydig cell development occurs.[62] The second time is two months post-partum, which leads to the proliferation of Leydig cells. However, there is subsequent atrophy of Leydig cells leading to the interstitium of the testis becoming inhabited by inactive precursor cells. Mature adult Leydig cells develop during puberty.[63] The third peak of testosterone is during the onset of puberty.

Leydig cell androgen production is dependent on the activity of four enzymes. These are cytochrome P450 17a-hydroxylase (P450 17a),  P450 side-chain cleavage enzyme (P450scc), 17b-hydroxysteroid dehydrogenase (17b HSD) and 3b-hydroxysteroid dehydrogenase (3b-HSD).[64] Leydig cells convert cholesterol into pregnenolone, and pregnenolone then converts into progesterone. Progesterone then metabolizes into testosterone. A mutation in the genes that code for the enzymes P450 17a, P450scc, or 3b-HSD can cause congenital adrenal hyperplasia, whereas a mutation in the gene for 17b-HSD causes 17b-HSD deficiency. These can all present with impaired virilization.

Exocrine Function

Spermatozoa are produced from spermatogonia through a stepwise process. The spermatogonia are at the basement membrane of the seminiferous tubules. The spermatogonia can be either stem cells (type A spermatogonia) or differentiated spermatogonia (Type B spermatogonia). Type A spermatogonia either produce more type A spermatogonia or type B spermatogonia.[65][66][67]  Type B spermatogonia divide and produce primary spermatocytes. The primary spermatocytes then enter prophase of meiosis 1. During this period, they move away from the basement membrane towards the lumen of the seminiferous tubule.

After completing the prophase of meiosis 1, the spermatocytes rapidly complete meiosis 1. This process results in the formation of haploid secondary spermatocytes. The secondary spermatocytes complete meiosis 2, leading to the production of four haploid spermatids from one primary spermatocyte.[55] Spermatids undergo spermiogenesis, the process during which the spermatids become spermatozoa and acquire the characteristic features of spermatozoa, such as the formation of a tail and loss of cytoplasm.[55] Sertoli cells, also known as ‘nursemaid cells,’ provide nutrients, growth factors, and organization to the developing spermatids.[68]

Functional Significance of Testicular Descent

According to the ‘temperature-dependent hypothesis,’ testicular descent is necessary because a low-temperature environment is needed to produce viable sperm. Normal spermatogenesis and storage in the epididymis require temperatures below that of the core body temperature.[69][70][71]

A testicular temperature of 2 to 7 degrees C below core body temperature is a requirement for effective spermatogenesis.[72][69] The testes have anatomical features that optimize temperature regulation. Thin scrotal skin with a large number of sweat glands, the pampiniform plexus which is a venous plexus surrounding the testes, the cremaster muscle which will contract or relax in response to temperature, the tunica dartos and a lack of adipose tissue also have an essential role in regulating the temperature.[69]

Imaging

In the context of undescended testis, imaging methods can serve to determine the anatomical location of undescended testes; this allows for better pre-surgical planning and reduces the extent of exploration and anesthesia time.[73] The magnetic resonance imagining (MRI) and the ultrasound (US) are useful in diagnosing and locating undescended testis. These techniques are preferable to others as they are non-invasive and do not use ionizing radiation. The diagnostic accuracy of MRI and US are similar to other methods.[74][75][74]

Karyotyping

Chromosomal changes are detectable through the use of a technique known as karyotyping, which allows the identification of the chromosomes so that in situations where there may be an extra or missing chromosome, it is detectable through karyotyping.[76] Karyotyping is a recommended investigation in all patients who present with disorders of sexual differentiation such as cryptorchidism and proximal hypospadias.[77]

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

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

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

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.

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

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.

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]

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 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.

Testicular Developmental Disorders

Cryptorchidism

Congenital undescended testes (cryptorchidism) is a very common congenital urogenital malformation with a prevalence among normal birth weight boys varying between 1.8 to 8.4%.[150] 

By definition, cryptorchidism is a condition where either one or both of the testes do not descend into the scrotum.[151] The undescended testes can be found in the usual route of descent or an abnormal ectopic position, leading to a way of classifying undescended testes according to their position:

• Cryptorchid – This refers to a testis that is not in the scrotum and cannot be manipulated so that they enter the scrotum.
• Ectopic – This refers to a testis that has not followed the regular developmental route of testicular descent. This condition can be femoral, pubopenile, crossed scrotal, or perineal.[152]
• Gliding – This refers to a testis that can be manipulated to enter into the upper scrotum, but with the removal of manipulation, they retract.[153]
• Retractile – This refers to the testis, which can be manipulated to enter into the scrotum; they do not retract when tension is released.
• Acquired – This refers to the testis, which initially had normal descent that ascends spontaneously.[154]

The diagnosis of cryptorchidism is by manual palpation; this is an essential part of the newborn physical examination and should be diagnosed during infancy.[155] Further investigation of undescended testicles includes various imaging techniques such as ultrasound, CT, and MRI.[73][156]

Anorchia

Anorchia occurs in 1/20,000 males and is also associated with 1/177 of those with cryptorchidism.[157][158] The majority of patients with anorchia have a normal phenotype, but some may present with abnormalities of the external genitalia.[159] The characteristic phenotype in a patient with anorchia is the absence of gonads despite having an XY karyotype. Most cases are associated with small tubular rudimentary Mullerian/Wolffian remnants. The phenotypical presentation can range from phenotypic females with a small, well-formed uterus to a phenotypical male with no testes.[160]

It is crucial to distinguish anorchia from cryptorchidism by examination of the abdomen and inguinal canal. Furthermore, as congenital adrenal hyperplasia can present similarly, plasma 17a-hydroxylase levels should be determined to rule out CAH. The diagnosis of anorchia can be made through undetectable plasma levels of AMH and inhibin B associated with raised plasma levels of FSH.[98] Testosterone therapy can be given to ensure the patient reaches a preferable height during puberty.

Monorchism

Clinically, it is not always possible to determine the exact etiology of monorchism as a child presenting with monorchism may have cryptorchidism, testicular agenesis, or VTS. Further investigation, such as laparoscopy or imaging techniques, may be performed. However, the accuracy of imaging techniques and the invasive nature of diagnostic laparoscopy means are not optimal methods for determining the cause of monorchism.[161] Alternative methods to determine the cause of monorchism is the volume of the contralateral testis in adults. However, in younger patients, this is not useful in predicting the cause of monorchism.[162][161] Fixation of the contralateral testicle is considered in children with monorchism to reduce the risk of torsion later in life to avoid a total loss of reproductive and endocrine function.[105]

Polyorchidism

The majority of patients with polyorchidism are asymptomatic.[163] It is usually a coincidental finding on the investigation of other symptoms such as hernia (30%), torsion (15%), mal-descent (40%), malignancy (6%), and hydrocele (9%).[164][165][166] In the majority of cases, the supernumerary testis is found on the left side [167]. The supernumerary testis may be found in the inguinal region (23%), scrotum (66%) or the abdomen (9%).[164][165]

Testicular Torsion

The prevalence of testicular torsion in males below the age of 25 years is between 2.9 to 3.8 patients per 100,000 people.[168][169][170] The classical clinical presentation of testicular torsion is acute severe scrotal pain, which occurs at rest, associated with some type of trauma. The severe pain can cause the patient to become nauseous or to vomit.[171] Testicular torsion tends to be associated with an absent cremasteric reflex.[171] Testicular torsion has a significant impact on the patient's future fertility and can be associated with psychological trauma.[172] Surgical intervention in testicular torsion has to be prompt. There is a 90 to 100% salvage rate if operation occurs within 6 hours of the onset of testicular torsion. The salvage rate reduces with time.[173]

Testicular torsion is diagnosable using a color doppler ultrasound; this is the gold standard for the evaluation of the acute scrotum.[174] Testicular torsion management involves emergency surgical exploration. Following this, the testis is untwisted and wrapped in a warm soaked gauze. The testis is then observed to see if there is an improvement in the color of the testis. To reduce the likelihood of the contralateral testis undergoing torsion, it is fixed along with the testis, which underwent torsion.[171]

Swyer Syndrome

Swyer syndrome has an incidence of 1 in 80,0000.[122] Swyer syndrome patients present with an above average height, underdeveloped breasts with normal axillary/pubic hair. The external genitalia is distinctly female; however, the uterus is small. The underdeveloped gonads are dysgenetic strips that do not have any endocrinological or reproductive function.[122] Patients with Swyer syndrome have an increased incidence of gonadal tumors such as gonadoblastomas or dysgerminomas.[122] One-third of patients with Swyer syndrome may suffer from dysgerminomas.[122]

Klinefelters Syndrome

In Klinefelter's syndrome, the primordial germ cells fail to develop appropriately such that by puberty, no germ cells remain, with resulting insufficient spermatogenesis.[175] Klinefelters syndrome is also associated with characteristic phenotypical features such as [125]:

• Small firm testes
• Above-average height
• Erectile issues
• Sparse male-pattern body hair
• Bilateral gynecomastia
• Infertility

5-ARD Deficiency

The phenotypical presentation of 5-ARD deficiency does not tend to match the genotypical presentation such that patients with the same genotype may present with different phenotypes.[176] However, phenotypical presentations include ambiguous genitalia with unfused labioscrotal folds resembling the labia majora or a phallus with a clitoris-like appearance.[177] Nevertheless, the internal genitalia such as the vas deferens, epididymis will be present. Undescended testes are also associated with 5-ARD deficiency.[177]

Congenital Adrenal Hyperplasia

A deficiency in 3B-HSD accounts for between 1% and 10% of CAH cases.[178][179] A deficiency in the P450 17a enzyme accounts for approximately 1% of CAH cases.[180] Deficiencies in the P50scc enzyme are incredibly rare and tend to present with a significant clinical variation.[146]