Development
In humans, biological sex is determined by the complement of sex chromosomes that provide instruction for an undifferentiated embryo to form along a male or female path. Initially, a conceptus is only sexually distinct by its karyotype, where males have XY and females have XX sex chromosomes. Sex differentiation is the subsequent dynamic and complex process regulated by various genetic and environmental causes that differentiate the indifferent gonad, internal genitalia, and external genitalia along a male or female path.
Gonadal Differentiation
Gonadal differentiation is the process that starts with an undifferentiated bipotential gonad and ends with its development into a testis or ovary. Specific genes induce gonadal differentiation, which begins around the fifth week of development post-fertilization with the formation of the gonadal primordium.[3][5][6] A thickened mesothelium develops medial to the mesonephros and proliferates to form the urogenital ridge on each side of the embryo. The paired urogenital ridges appear as longitudinal ridges in the fourth week post-fertilization and are composed of intermediate mesoderm covered by coelomic epithelium.
The urogenital ridges are common precursors that contribute to the formation of the urinary system, genital system, and adrenal cortex. The urogenital ridge divides into a urinary and an adreno-gonadal component. For the latter, specialized cells form in close proximity to territory, forming the gonadal ridge and ultimately contribute to the formation of steroidogenic cells of the adrenal gonads and adrenal glands. Additionally, finger-like epithelial cords, the primitive sex or gonadal cords, develop in the gonadal primordium and grow into the mesenchyme. At this point in development, the indifferent but bipotential gonads are composed of an outer cortex and inner medulla with different fates in male and female gonadal differentiation. In both XX and XY embryos, the primordial germ cells migrate from the umbilical vesicle along the dorsal mesentery to reach the region of the gonadal ridges and are incorporated into the gonadal cords. The primordial germ cells will ultimately form oocytes and sperms later in development.
Testis
If an embryo is chromosomally male (46 XY), the bipotential gonad differentiates into testes under the influence of the SRY gene. The SRY gene is a single exon gene, and it encodes a transcription factor called sex-determining region Y protein (SRY protein), also known as the testis-determining factor (TDF). SRY protein, or TDF, is a sex determination gene that directs male gonadal differentiation. SRY gene expression aids in differentiating Sertoli cells, which later results in the production of Müllerian inhibiting substance (MIS) or anti-Müllerian hormone (AMH). SOX9 also helps in Sertoli cell differentiation, which is upregulated by the SRY gene. It is a critical step in the initiation of testis development.[7]
Early in male gonadal development, TDF induces the gonadal cords to condense and anastomotic channels to form the rete testis within the medulla of the indifferent gonad. The tunica albuginea forms and separates the gonadal, or primitive sex, cords from the surface epithelium and is an observable indicator of the development of testes. The gonadal cords are referred to as seminiferous cords after the development of the tunica albuginea and eventually form the seminiferous tubules, straight tubules, and rete testis. The seminiferous tubules are separated from each other by mesenchyme that forms interstitial tissue composed of interstitial or Leydig cells. The seminiferous cords (and later tubules) are composed of sustentacular cells, or Sertoli cells and their walls are formed of spermatagonia and Sertoli cells. The interstitial or Leydig cells secrete testosterone by eight weeks of development, which is critical for the male sexual differentiation of the internal and external genitalia. Around the same time, the Sertoli cells of the seminiferous tubules produce Müllerian inhibiting substance (MIS) or anti-Müllerian hormone (AMH).
The vascularization of the gonadal ridge is a dynamic process. The developing testis recruits and patterns vasculature by a remodeling mechanism, whereas the developing ovary recruits vasculature by normal angiogenesis. In developing testes, pre-existing mesonephric vessels dissociate and form a cluster of endothelial cells that migrate and eventually underly the coelomic epithelium of the gonad. Here, the endothelial cells assemble to form the coelomic vessel, which extends along the length of the testes at its anti-mesonephric margin. The formation of the coelomic vessel is one of the earliest hallmarks of testes development.
The testes start to descend from the abdominal cavity at only ten weeks of development. The descent is aided by the gubernaculum, a fold of peritoneum attached to the testis. The descent of the testes is completed by the 25th to 35th weeks of development.[8][9][10] Undescended testes are more common in premature and low birth weight infants. Those with undescended testes have an increased chance of getting testicular tumors, especially germ cell tumors.
The mesonephric ducts form the main genital duct of the male embryo, the ductus deferens, as described below. The remaining parts of the excretory tubules form the efferent ductules, which make the rete testis continuous with the ductus deferens. The seminiferous tubules and rete testis tubules enter into the ductules efferents.[11][1]
Ovary
In a fetus with XX chromosomes, the gonads are initially indifferent and remain indifferent longer than in male gonadal development. Both autosomal genes and genes on the X chromosome contribute to ovary formation. Ovary-specific transcription factors induce ovarian differentiation; these include FOXL2 (forkhead transcription factor 2), WNT4 (wingless-type MMTV integration site family member 4), RSPO1 (R-spondin 1), and the beta-catenin pathway.[11] Only recently has the differentiation of the ovary been identified as an active and complex process.[12] It is now understood that the absence of an active SRY gene in a chromosomal male will not result in ovarian differentiation but in variable gonadal dysgenesis.[12]
A chromosomally female fetus (46 XX) will not have a histologically distinct ovary until approximately 10 weeks post-fertilization.[12] The gonadal cords will grow into the medulla of the developing ovary to form a network of canals, the rete ovarii. Both the canals and the gonadal cords eventually degenerate, leaving irregular cell clusters in the medullary part of the ovary. Later, these irregular cell clusters are replaced by vascular stroma that form the ovarian medulla.
In contrast to the gonadal cords, cortical cords arise from the surface epithelium of the developing ovary, grow into the underlying mesenchyme, and eventually house the primordial germ cells. Primordial germ cells increase in number in the fetus through the process of mitosis, and they eventually differentiate into oogonia.[12] Oogonia do not form post-birth. In the 10th week, oogonia in the center of the ovary enter meiotic prophase as the first obvious indicator of morphological ovarian differentiation. The maturation of the ovary proceeds in this manner from the center to the periphery.[12]
Oogonia become enveloped by a single layer of follicular, or granulosa, cells. By the end of the 7th month of gestation, mitotic activity has stopped, and almost every germ cell has entered meiotic prophase I and is arrested in the diplotene stage—primordial follicles house the oogonia arrested in meiosis (the first stage of folliculogenesis). The primordial follicles remain dormant until they are stimulated at sexual maturity to form primary follicles (the second stage of folliculogenesis).[13] After sexual maturity, a small sample of primary oocytes mature monthly and complete the first meiotic division to form a secondary oocyte. The process involving long-term dormancy and completion of meiosis at ovulation is distinct in females, whereas meiosis begins at puberty in the male gonad.
Another notable distinction between male and female gonadal development involves the role of the germ cells. Fetal testis development is not dependent on germ cells, while ovarian follicles require germ cells for their development. So the involvement of germ cells in the stabilization of gonads is one of the major differences between the development of the ovary and testis. The characteristic feature of testis differentiation is the formation of the coelomic vessel, which does not occur in the development of the ovary.[11][14][12]
Differentiation of the Internal Genitalia
Early in development, both XX and XY embryos have two pairs of genital ducts: the paramesonephric ducts (or Müllerian ducts) and mesonephric ducts (or Wolffian ducts). Both pairs of genital ducts form on the surface of the mesonephric kidneys. Around 6 to 7 weeks post-fertilization, the paramesonephric ducts develop adjacent to the mesonephric ducts. At first, both pairs of ducts are indifferent. In sexual differentiation along a female line, the paramesonephric or Müllerian ducts play a major role in developing the female internal genitalia. The Müllerian ducts form the fallopian tubes, uterus, and superior portion of the vagina. In chromosomal males, the Müllerian ducts typically regress under the influence of testis-derived Müllerian inhibiting substance, also known as anti-Müllerian hormone.[15] In males, the Wolffian ducts are critical to the formation of the male internal genitalia and produce the epididymis, vas deferens, and seminal vesicle. The male differentiation of the Wolffian ducts is mediated by testis-derived androgens. In females, the Wolffian ducts regress.
Müllerian Ducts
The Müllerian ducts are the progenitor of the upper female genital tract, and they initially develop from intermediate mesoderm. Müllerian duct formation involves three phases: specification, invagination, and elongation.[16] The Müllerian ducts first develop as a coelomic epithelial cleft on the surface of the gonadal ridges at 5 to 6 weeks post-fertilization. Ultimately, the coelomic invaginations form the infundibular openings of the fallopian tubes in the peritoneal cavity. The paired Müllerian ducts grow caudally to reach the urogenital sinus. If the Müllerian ducts fail to merge with the urogenital sinus, lower vaginal agenesis may result. Sometimes failure in the formation of the Müllerian duct results in uterine and vaginal agenesis in females, leading to primary amenorrhea in girls (MRKH syndrome). The Müllerian ducts form fallopian tubes at their cranial ends and fuse along the midline to form the uterus and upper aspect of the vagina at their caudal ends.
Early in the fetal development of a male, the Sertoli cells of the testes begin to secrete anti-Müllerian hormone. lt is mediated by beta-catenin, causing the Müllerian ducts to regress instead of developing into female reproductive organs.[15][16][17][18]
Wolffian Ducts
The Wolffian ducts, or initially mesonephric ducts, arise from the intermediate mesoderm. The mesonephric ducts play a significant role in the developing fetus of both males and females. These ducts induce the formation of three successive generations of kidneys: the pronephros, mesonephros, and metanephros. Initially, the mesonephric ducts drain the first-generation kidney or pronephros. Later in development, the mesonephric duct is incorporated into and functions as the excretory duct of the second-generation kidney, or mesonephros. After the development of a definitive kidney, the remaining mesonephric ducts acquire a reproductive developmental role and are often referred to as Wolffian ducts in this context.[19][20]
In males, the cranial portions of the Wolffian ducts persist and form the appendix epididymis and the rete testis. The part of the Wolffian duct just inferior to this area elongates and convolutes to form the epididymis. The remainder of the Wolffian duct gradually develops a thick muscular coat and forms the ductus (or vas) deferens. The Wolffian ducts also produce the seminal vesicles, which develop from the caudal end of the ducts as a lateral diverticulum. In addition, a part of the Wolffian duct forms the common ejaculatory duct between the seminal vesicle and urethra.
During the 9th and 10th weeks post-fertilization, SRY-induced Leydig cell differentiation occurs and leads to testosterone production. Testosterone plays an important role in the stabilization and male differentiation of the Wolffian duct. Locally produced testosterone from the testis is essential for the direct virilization of the Wolffian ducts. The absence of testosterone in females, and some males, leads to regression of the Wolffian duct during sexual differentiation.[21][22][23]
Differentiation of the External Genitalia
Just like the gonads and internal genitalia, the external genitalia is indifferent in early development. In the 5th week, mesenchymal cells migrate to the perineum to form swellings on either side of the cloaca called cloacal folds. On the cranial aspect of the cloacal folds, the mesenchymal cells fuse along the midline to form the genital tubercle. The genital tubercle is located just above the urogenital ostium, and it is the primordium of the future developing clitoris or penis. Quickly after the development of the genital tubercle, urogenital and labioscrotal folds form lateral to the cloacal membrane. These stages are identical in chromosomally male and female fetuses, and they are identical until around the 9th week post-fertilization.[8] At this point, the genital tubercle undergoes elongation and sex differentiation along the path leading to either the primordial male penis or primordial female clitoris.
Male External Genitalia
In male embryos, masculinization of the external genitalia in utero occurs in response to dihydrotestosterone (DHT).[20] Leydig cell-secreted testosterone is converted into DHT by 5-alpha-reductase. In response, the genital tubercle undergoes growth and elongation to form the penis—the scrotum forms by the fusion of the labioscrotal folds at the midline. Later in development, the fusion site is demarcated by the scrotal raphe. A solid epithelial, or urethral, plate forms on the ventral surface of the developing penis. The epithelial plate undergoes canalization by first forming a groove on the surface of the genital tubercle, which is bounded by the urethral folds. Then, the urethral folds fuse along the midline and convert the urethral groove into a fully contained penile urethra, where the site of fusion of the surface ectoderm is demarcated by the penile raphe. Failure of this process may result in the formation of hypospadias due to inadequate androgens during this critical developmental period. Hypospadias is an abnormal location of the external urethral meatus somewhere along the ventral surface of the penis and is named based on its site: glanular, coronal, penile, or perineal.
The glans penis forms an ectodermal cord that extends proximally to become continuous with the spongy urethra. The penile prepuce forms around 12 weeks post-fertilization due to ingrowth of ectoderm near the margin of the glans penis that ultimately breaks down. The development of the male external genitalia is typically complete by 14 weeks of development.
Female External Genitalia
Female external genital development is regulated by the absence of androgens and the presence of maternal estrogens. In the absence of testosterone, the genital tubercle enlarges to form the clitoris but at a relatively slower rate than genital tubercle enlargement in males. Female external genital development is distinct from male development in that the urogenital and labioscrotal folds remain largely unfused. The urogenital folds fuse along their posterior margin to form the frenulum of the clitoris, while the unfused aspects of the urogenital folds form the labia minora. The labioscrotal folds fuse anteriorly to produce the anterior labial commissure and the mons pubis, and they fuse posteriorly to form the posterior labial commissure. However, the majority of the labioscrotal folds remain unfused and ultimately form the labia major.
Vaginal development involves both the Müllerian ducts and the urogenital sinus. The caudal end of the fused Müllerian ducts forms the uterovaginal primordium that contacts the posterior part of the urogenital sinus. This process results in the formation of the sinus tubercle, which induces paired endodermal outgrowths to form sinovaginal bulbs. The sinovaginal bulbs connect the urogenital sinus and the uterovaginal primordium. The sinovaginal bulbs fuse to form a solid vaginal plate that later forms an open lumen. The sinovaginal bulbs produce the inferior two-thirds of the vaginal canal. The hymen is produced during uterovaginal development as a lining that separates the vagina canal from the urogenital sinus.
The epithelium of the vagina is endodermally derived, while the muscular walls of the vagina are mesenchymal derived. The differentiation of female external genitalia starts at 11 weeks and completes by 20 weeks post-fertilization.[8]
Clinical Significance
Careful examination of a neonate is critical and includes evaluating the symmetry and pigmentation of the external genitalia, presence of palpable gonads, and labioscrotal morphology. In addition, measurement of the phallus should be recorded, and the position of the meatal opening and number of perineal openings assessed. After birth, biological sex assignment is an important clinical decision. Biological sex is often assigned at the time of birth based on the appearance of the external genitalia. Examination of the external genitalia is the simplest way to assign sex post-birth but is less reliable than genetic evaluation. The external genitalia is highly prone to variation from sexual differentiation along a typical male or typical female pathway and may present with some degree of ambiguous genitalia. Neonates presenting with any degree of ambiguous genitalia tend to provide a challenge to doctors who manage their care and cause distress to their parents.[12]
Differences of sex development (DSDs), previously known as disorders of sex development or intersex conditions, are a group of conditions associated with atypical development of the gonads, internal genitalia, or external genitalia. In some individuals, there is a conflict between chromosomal sex and sexual differentiation of the gonads, external genitalia, internal genitalia, or (later) secondary sex characteristics. DSDs are sometimes identified in utero due to routine genetic evaluation, at birth due to the presence of ambiguous genitalia, or sometimes well after birth due to virilization, delayed puberty, or infertility.[12] Patients with these conditions are reported to face the stigma that negatively impacts their psychosocial health.
Differences in sex development can be organized along wide-ranging classification schemes. There are numerous conditions that are classified as DSDs; some of the more common or better understood include:[24][12]
46 XX DSD
This group of DSD conditions impacts individuals with XX sex chromosomes with atypical development of the gonads, internal genitalia, external genitalia, and/or secondary sex characteristics. Some of these conditions include:
Aberrant Ovarian Development
Androgen excess
Fetal (21- or 11-hydroxylase deficiency)
Fetoplacental (aromatase deficiency)
Maternal (luteoma, exogenous)
46 XY DSD
This group of DSD conditions impacts individuals with XY sex chromosomes with atypical development of the gonads, internal genitalia, external genitalia, and/or secondary sex characteristics. Some of these conditions include:
Abnormal Testicular Development
Defects in Androgen Biosynthesis
Defects in Androgen Action
Disorders of AMH and AMH Receptor
LH Receptor Defect
Others (hypospadias and cloacal exstrophy)
Sex Chromosome DSD
This group of DSD conditions impacts individuals with atypical sex chromosomes that result in a potential suite of developmental outcomes. Some of these conditions include:
45 X (Turner syndrome and its variants)
47 XXY (Klinefelter syndrome and its variants)
Ovotesticular DSD
Discussion
Congenital adrenal hyperplasia (or CAH) is a common cause of 46 XX DSD resulting from excess androgens. This is a group of disorders that impact the development of the adrenal glands and may involve several metabolic effects. In most individuals with this condition, there is a 21-hydroxylase enzyme deficiency that results in mineralocorticoid deficiency and excessive formation of androgenic products. This condition causes excess virilization of an individual with XX sex chromosomes. The clinical presentation is dependent on the severity of excess androgens and the timing of development.
Androgen insensitivity syndrome (AIS), or complete androgen insensitivity, is a condition in which a person with XY sex chromosomes does not respond to androgens. Individuals with this condition, while chromosomally male, present at birth and through development as female with typical female psychosexual characteristics and are often raised as female. These individuals have typical female external genitalia but no uterus and are thus infertile.
Partial androgen insensitivity is a similar 46 XY condition involving under-masculinized morphology. Individuals with partial androgen insensitivity syndrome may have mildly virilized female external genitalia (clitoromegaly) to mildly under-virilized male type external genitalia (hypospadias and/or microphallus). In both cases, the affected individuals have testes and typically have normal production of testosterone and DHT.
An autosomal recessive 46 XY DSD, 5-alpha-reductase type 2 enzyme deficiency results from a mutation in the SRD5A2 gene. This enzyme converts testosterone to DHT, which is responsible for the masculinization of external genitalia in utero. Neonates with 5-alpha-reductase deficiency present with variable degrees of ambiguous genitalia, such as clitoral-like phallus, bifid scrotum, and pseudovaginal perineoscrotal hypospadias. At puberty, since masculinization at this time involves other androgens, the afflicted individual may have increasingly virilized external genitalia.
Hypospadias is an example of a common 46 XY DSD condition that involves an ectopic external urethral meatus on the ventral penis. Depending on the location of the external urethral meatus, the hypospadias may be asymptomatic or may cause additional challenges. For instance, granular hypospadias may be an incidental finding, while penile or perineal hypospadias may be associated with abnormal spraying of urine, abnormal curvature of the penis (chordee), or infertility via sexual intercourse. Epispadias are distinct from hypospadias as they tend to occur much earlier in development and present as an abnormal opening of the external urethral meatus on the dorsal aspect of the penis.
Sex chromosome DSDs include such conditions as Turner syndrome and Klinefelter syndrome. The incidence of Turner syndrome is approximately 1 in 2500 liveborn females. Turner syndrome is often diagnosable at birth due to features like low birth weight, lymphedema of hands and feet, and short neck. However, other forms of Turner syndrome may present later with short stature and delay in puberty. Some Turner syndrome karyotypes include 45 XO, 45 XO/46 XX, or 45 XO/ 46 XY. Klinefelter syndrome is another sex chromosome DSD that occurs in 1 in 1000 liveborn males. Clinical features of affected individuals include tall stature, weak muscles, small testes, gynecomastia, delayed puberty, and infertility.[24][25][26][27]
