Female Genital Tract Development and Disorders of Childhood

The Genital Ridge

The genitourinary system begins to develop by the fifth week postfertilization as a longitudinal ridge of undifferentiated mesenchymal cells that extend bilaterally, flanking the mesenteric root (see Fig. 1.2 ). Excluding the bladder and external genitalia, the remainder of the genitourinary system ultimately evolves from this mesenchymal thickening. The undifferentiated mesenchyme in this area comprises the genital ridge and will ultimately give rise to the medulla of the ovary, whereas the coelomic epithelium becomes the ovarian cortex and ovarian surface epithelium (see Fig. 1.3 ). Genital ridge development is under the control of homeobox gene family members, transcription factors, and wingless family signaling factors that are integral to genital ridge development, and mouse knockouts affecting these genes will nullify genital ridge development and that of the adjacent kidneys and adrenals. In particular, several genes ( LHX1 [LIM1], EMX2, PAX2, WNT4, WNT7a, HNF1β [TCF2], and DACH1/2 ) are critical to the formation of the urogenital anlagen before sexual differentiation (see Fig. 1.4 ).

Ovary Development and Sex Determination

Human germ cells enter the genital ridges between 4 and 6 weeks' gestation. In the presence of a male genotype containing the sex-determining region gene (SRY) on the Y chromosome, or in the event of an XX genotype in which the SRY region has been retained via translocation, the embryo will develop into a male. The primary target of SRY is SOX9 , a homeobox gene that has been shown to “rescue” the male phenotype in some XX individuals. In the absence of SRY , granulosa cells form single layers that invest the primitive oocytes. These primordial follicles rapidly multiply to more than 7 million by the 22nd week. At this point, cell division ceases and the cell population drops by more than two-thirds at birth and by another 90% by puberty, when the average number of oocytes in the ovary is approximately 300,000. In the genetically female fetus, the gonad is distinguished by the end of the second month due to estradiol production from the ovarian stroma. The primitive germ cells proliferate and differentiate to oogonia, beginning in the center of the ovary and moving toward the periphery over time. The oogonia become invested with a single layer of follicular cells derived from coelomic epithelium of mesonephric origin, become oocytes, and form primordial follicles ( Fig. 1.5 ). The earliest follicles develop by 15 weeks. By the end of the seventh month of gestation, all the germ cells have ceased to proliferate and have entered meiotic prophase, where they will remain until ovulation. In contrast to the testis, germ cells in the ovary are critical to the development of their supporting stromal cells. In their absence, the prefollicular cells are not sustained, and a streak gonad will eventuate.

Germ cells exhibit brisk proliferative activity in weeks 15 to 20, with high levels of Ki-67 expression in the cortex and medulla ( Fig. 1.6A ). Concurrent with proliferation is the expression of OCT4, a transcription factor that is expressed early in embryogenesis and has been demonstrated to be integral to maintaining viability of the primordial germ cell mass (see Fig. 1.6B ). OCT4 staining is concentrated primarily along the outer rim of the primitive ovary at this point. In the center, a gradually increasing population of enlarged oocytes is seen, and these cells display strong nuclear staining for p63 (see Fig. 1.6C ). Between week 20 and term, the percentage of germ cells staining for OCT4 progressively declines at the periphery and, as germ cells decline in number, a progressively increasing proportion become p63-positive. Postnatally, all identifiable oocytes show intense p63 nuclear positivity (see Fig. 1.6D ).

This sequence of immunostaining patterns is consistent with the role of OCT4 in maintaining primordial germ cells through the proliferative phase in the first two trimesters of pregnancy. Unknown factors result in the programmed cell death of a large number of the remaining germ cells in the last trimester. The preservation of a discrete subset is coincident with the expression of p63, which is expressed subsequently throughout the life of the oocyte in the ovarian cortex. Studies in mice have identified p63, and specifically the TA isoform, as the “guardian of the female germ line,” required for the process of cell death induced by DNA damage, similar to the function served by p53 in somatic cells.

The preceding process is under control of several genes at critical points. Expression of LHX9 appears to be necessary for the development of supporting ovarian stroma. In mouse models lacking this gene, germ cell migration is normal, but the somatic cells of the genital ridge fail to proliferate, with failure of gonad formation. LHX9 mutants do not display other disorders, making this gene an attractive candidate for isolated gonadal dysgenesis. However, mutations in this gene have yet to be identified in humans. Mutations in another gene, CBX2, have been identified in 46,XY females with normal genitalia and ovaries. Disruption of expression of CBX2, a component of the Polycomb group complex of regulatory proteins, leads to delayed development of the genital ridge and male to female sex reversal. This phenotype has been linked to CBX2 's regulation of Sry gene expression. Yet another gene, GATA4, has also been linked to disorders of gonadal development in human. Unlike other genes involved in growth and maintenance of the genital ridge, this gene has been implicated in gonadal initiation as the coelomic epithelium in the GATA4 -conditional knockout mouse fails to thicken, remaining as a morphologically undifferentiated monolayer.

Maintenance of ovarian germ cells is dependent on an intact X chromosome. X chromosomal abnormalities, such as monosomy X, are associated with accelerated follicular atresia. These include deletions of Xp11, Xq13, and specific genes such as ZFXA and DAZLA.

The Uterus and Vagina

Concurrent with genital ridge formation and germ cell migration during the fourth postfertilization week is invagination and tubal extension of coelomic epithelium in the transition area between the pronephros and mesonephros to form the paramesonephric (müllerian) ducts. Rostrally, the paramesonephric ducts lie lateral to the mesonephric (Wolffian) ducts; caudally, their paths cross the mesonephric ducts ventrolaterally to meet in the midline, where they fuse by the eighth week of gestation. It is now believed that the müllerian duct grows by active epithelial proliferation using the mesonephric ducts as a guide. The extracellular signaling molecule Wnt9b is required for müllerian duct elongation, presumably because the guiding mesonephric ducts fail to develop in Wnt9b ^−/− embryos. LHX1 is the first transcription factor to be identified as essential in epithelial progenitor cells of the müllerian duct, with its lack of expression leading to uterine hypoplasia and complete loss of the endometrium.

Following midline fusion, the müllerian ducts merge and cavitate to form the uterovaginal cavity, which is destined to form the uterus and upper third of the vaginal canal. The more caudal portion remains solid and merges with an ingrowth of solid endoderm from the urogenital sinus, called the sinovaginal bulb. These solid formations undergo canalization by the 20th week to form the vagina, with the hymen demarcating the junction of paramesonephric (cephalad) and endodermal (caudad) tissue origins. The upper vagina, which is derived from the distal end of the müllerian duct, is flanked at its superior pole by recesses of mesonephric duct origin, which will eventually form the vaginal fornices ( Fig. 1.7 ).

The eventual epithelialization of the vagina and cervix is potentially explicable by two theories. The first resolves the presence of squamous mucosa by the ingrowth of squamous epithelium from the introitus. The second holds that the squamous mucosa of the vagina and cervix emerges via induction of basal cells in the müllerian epithelium of the cervix and vagina. This latter hypothesis is supported by several lines of evidence. First, p63 expression is seen along the full length of not only the vagina but also the urethra during this interval, indicating that urothelium and squamous epithelium develop via the same process ( Fig. 1.8 ). Second, although the newborn human vagina is fully covered by squamous epithelium, the vagina of the newborn mouse is lined by mucus-secreting, endocervical-type columnar epithelium, beneath which lies a single row of reserve cells. With the onset of estrus, the reserve cells expand and undergo squamous differentiation, after which the vagina is permanently lined by mature squamous epithelium. This process of basal cell induction fails in the p63 null mouse, and the vagina remains lined by a combination of müllerian and primitive urogenital sinus epithelium.

The final development of the müllerian duct can be divided into three separate phases: (1) regional specification of tissue and organ identity; (2) partitioning and expansion of distinct endometrial and myometrial compartments; and (3) endometrial adenogenesis. Several of the homeobox gene family members have been identified as genetic factors required for assignment of tissue and organ identity along the length of the müllerian duct. From such studies, an idealization of the plan for segmentalization can be inferred. In such schema, HOXA9 expression defines the future oviduct. HOXA10 (rostral) and HOXA11 (caudal) expression is needed for uterine fundal development, and HOXA11 also is needed for cervical development. Finally, HOXA13 is expressed in the cervical and upper vaginal anlage. In null mutants, their patterns of segmental or partially overlapping expression along the müllerian duct (from the rostral to caudal ends) is disturbed and results in homeotic transformations. The downstream details of HOX gene activity on regionalization of the müllerian duct have yet to be elucidated. In addition, the developmental genetics of endomyometrial partitioning and endometrial adenogenesis have not been defined, in part reflecting the differences between the human female genital tract and those of lower, more commonly studied mammals.

The External Genitalia

The hindgut and urogenital sinus open into a common cloaca prior to the seventh week of gestation. At this point, a ridge of mesenchyme—the urorectal septum—migrates caudad toward the cloacal membrane and separates the genitourinary system from the rectum. An external midline protuberance develops at this point, the genital tubercle, which is flanked dorsolaterally by the genital folds, which are in turn laterally cuffed by the labioscrotal swellings. The genital tubercle ultimately becomes the clitoris, the genital folds eventually form the labia minora, and the labioscrotal swellings differentiate into the labia majora ( Fig. 1.9 ).

The factors involved in the modeling of the external genitalia are multiple and involve sequential expression of regulatory genes and the induction of gene expression by specific epithelial-stromal interactions. The sonic hedgehog gene (SHH) has been shown to regulate genes expressed in mesenchyme, including patched 1 (PTCH1), bone morphogenetic protein-4 (BMP4) , HOXD13, and fibroblastic growth factor (FGF10) . The absence of SHH (in SHH null mice) is associated with agenesis of the genital tubercle, accelerated cell death, diminished cell growth, and an abnormal shift in expression of BMP4 from the mesenchyme to the epithelium. Thus, SHH is considered to increase outgrowth and differentiation of the genital tubercle. Predictably, maintenance of the mucocutaneous genital mucosal epithelium is critical to the development of the appropriate mesenchymal response; p63 null mice, which are devoid of skin and squamous mucosa, fail to complete the urorectal septum, exhibit abnormalities in bladder and phallic development, and are born with a common cloaca. Humans with p63 mutations have defects in the growth of hair, teeth, and distal limb development. They exhibit less conspicuous genital anomalies but exhibit genital hypoplasia similar to that seen in the external genitalia of the p63 null mouse. The conclusion from study of these models is that the integrity of the squamous mucosa is also critical for normal caudal, urogenital, mesenchymal development.

The female genital phenotype was once considered a default phenotype, resulting from the absence of interference by androgenic hormones, despite inactivated estrogen receptor proteins or aromatases. SRY , the sex-determining gene located on the short arm of the Y chromosome, is critical in gonadal sex differentiation, such that the absence of SRY confers (or permits) female gonadal differentiation. However, current evidence also indicates that generation of the female phenotype occurs actively, via autosomal genes such as WNT4 on chromosome 1p, which functions along the pathway of DAX1 , an X chromosome gene, to antagonize SRY expression. Alternative pathways independent of genetic gonadal regulation, as with in utero diethylstilbestrol (DES) exposure, lead to inappropriate expression of estrogens, resulting in vaginal adenosis and structural malformations of the uterus and cervix.

The Ovary and Fallopian Tube

Developmental Abnormalities

Developmental abnormalities of the fallopian tubes and ovaries arise via three mechanisms: (1) disturbance in müllerian duct development; (2) disturbance in gonadal development; and (3) abnormal sex chromosomes.

Ovarian Hypoplasia

Pathogenesis

The classic example of ovarian hypoplasia is associated with Turner syndrome (45,X karyotype, also designated monosomy X). The primordial germ cells make their way to the genital ridge but fail to induce follicle development and degenerate postnatally, producing an ovary with no germ cells by toddlerhood. Because the Y chromosome is absent, normal genitouterine development takes place. However, because the ovary is not capable of promoting folliculogenesis and producing estrogens, external sexual characteristics remain infantile. Patients with a pure 45,X genotype are not at increased risk for gonadal neoplasia; it is the phenotypic female Turner syndrome patients with a Y chromosome constitution harboring the SRY gene who are at risk for gonadal neoplasia—namely, gonadoblastoma and dysgerminoma (seminoma).

Histopathology

The typical ovary in monosomy X is a streak gonad. Grossly, the ovary is a small, flat, ovoid structure; microscopically it consists principally of scant ovarian cortical stroma, which may contain scattered primitive sex cord structures. Oocytes are not present ( Fig. 1.10 ).

Agenesis and Dysgenesis of the Ovary

Pathogenesis

This disorder is not associated with a chromosomal anomaly; patients are therefore karyotypically normal (46,XX). The primordial germ cells neither develop nor migrate; therefore, the gonad does not develop. Maternal - placental estrogens complete development of the external genitalia and the müllerian ducts, but the external phenotypic stigmata of Turner syndrome are not seen in such cases.

Histopathology

The ovaries are streak gonads, indistinguishable from the Turner ovarian histologic phenotype.

Androgen Insensitivity (Testicular Feminization) Syndrome

Pathogenesis

These patients have a normal 46,XY male karyotype, do not respond to testosterone, and therefore are externally phenotypic females. Internally, the paramesonephric duct system is suppressed, uterine development is blunted or absent, and the vagina ends in a blind pouch. The testes do not descend and remain in the abdomen or inguinal region, where they are at risk of germ cell tumorigenesis, a risk that increases over time.

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