Female sexual development

Female development does not require the presence of a gonad, and the ovary plays no part in primary sexual development. This means that a neuter embryo will always develop along female lines. The testis-determining factor is the SRY gene carried in the sex-determining region of the Y chromosome. The indifferent gonad develops into an ovary when no Y chromosome is present, although two functional X chromosomes are usually required for normal ovarian differentiation. Disorders of female sexual development are listed in Table 19.1.

Table 19.1 Abnormalities of female sexual development

∗ Diagnosis of a specific type of intersex requires histological confirmation of gonadal status; ovotestis can look macroscopically exactly like a normal ovary, or the patient could have one macroscopically normal testis on one side and an ovary on the other.

Embryological development

Germ cells arise in the wall of the yolk sac, and migrate to the region of the coelomic germinal epithelium. In the sixth week cords of cells appear within the indifferent gonad, but it is not until after the seventh week that ovarian differentiation is apparent and by 14 weeks these cell cords surround the primordial follicles.

The paired paramesonephric Müllerian ducts arise as an invagination of the coelomic epithelium of the mesonephric ridge lateral to the mesonephric duct. The paramesonephric duct follows the mesonephric duct. Near the cloaca, the paramesonephric ducts cross from the lateral to the medial side of the mesonephric ducts (Fig. 19.1A); together they carry with them some mesoderm from the side walls of the pelvis to create the transverse bar which helps to form the septum dividing the rectum from the urogenital sinus.

Fig. 19.1 Development of the female genital tract. Frontal view of the posterior wall of 7-week embryo showing the mesonephric and paramesonephric ducts during the indifferent stage of development. Female genital tract in a newborn infant.

At the 30-mm stage (8 weeks), fusion of the parameso-nephric ducts creates the utero-vaginal canal, which ultimately forms the uterus and proximal part of the vagina (Fig. 19.1B); the unfused parts form the uterine tubes. The trans-pelvic bar, which is a continuation of the mesonephric mesentery, forms the broad ligament; the ovary, projecting medially from the mesonephric ridge in the early stage, comes to lie posterior to the broad ligament. The inferior free end of the fused paramesonephric ducts (utero-vaginal canal) is still solid, and the sino-vaginal bulbs grow out from the posterior wall of the urogenital sinus to fuse with it and, later, give rise to the lower part of the vagina. The hymen occupies the position where the sino-vaginal bulb and urogenital sinus meet. The gonads are at first elongated and lie in the long axis of the embryo. Later, each gonad assumes a transverse lie. The gubernaculum is formed in the inguinal fold as a fibromuscular band which burrows from the gonad to gain attachment to the genital swelling; thus the caudal pole of the gonad becomes relatively fixed. The gubernaculum persists as the round ligament of the uterus. The ovaries retain attachment to the posterior aspect of the broad ligament. The genital swellings form the labia majora, the genital folds form the labia minora, and the genital tubercle forms the clitoris.

Herpes virus infection

Sexually transmitted herpes virus infection is usually due to herpes simplex type 2, and produces painful ulceration of the vulval skin. Histologically, intra-epithelial blisters are seen, accompanied by specific cytopathic effects characterised by intranuclear viral inclusions and eosinophilic cytoplasmic swelling.

Syphilis

Primary and secondary syphilitic lesions may affect the vulva. The condyloma latum of secondary syphilis is characterised by oedematous, acutely inflamed, hyperplastic epithelium with underlying chronic inflammation, including prominent plasma cells. Hypertrophy of endothelial cells (‘endarteritis obliterans’) may occur. A silver stain may be used to demonstrate spirochaetes.

Granuloma inguinale (donovanosis)

This sexually transmitted disease caused by the Gram-negative bacillus Calymmatobacterium granulomatis is found throughout the tropics. It commonly causes chronic vulval ulceration but may spread to other sites in the female genital tract. Characteristically, large macrophages with clear or foamy cytoplasm containing bacilli (Donovan bodies) are seen, demonstrated with the help of Giemsa or silver stains.

Lymphogranuloma venereum

This sexually transmitted disease is caused by Chlamydia trachomatis and is most prevalent in the tropics. The earliest lesion is a vesicle which breaks down to form a punched-out painless ulcer. The primary lesion may become secondarily infected with abundant granulation tissue, fibrosis, fistula formation and lymphatic obstruction characterising the chronic form of the disease. Necrotising granulomas may occur in inguinal lymph nodes.

Candidiasis

Candida may cause chronic irritation and inflammation of the vulva that may be associated with vaginitis. The diagnosis may be made by microscopic examination of skin scrapings or culture. The histological features are non-specific, although the fungi may be identified within the keratin layer or superficial epithelium with the use of silver stains.

Cysts and tumours

Any benign cyst or tumour of the skin may be seen in the vulva. Two uncommon benign tumours are worthy of comment because of their distinct histological appearance: papillary hidradenoma and granular cell tumour.

Bartholin’s glands

Bartholin’s glands are common sites of cysts and of abscesses secondary to infection of a cyst. Bartholin’s gland adenoma is uncommon, and adenocarcinoma arising at this site is rare.

Intra-epithelial neoplasia

The term intra-epithelial neoplasia refers to the spectrum of pre-invasive neoplastic change affecting the vulva. Its classification is the same as that of similar lesions in the cervix, although it may be incorrect to draw too close an analogy with the cervix as far as natural history is concerned. It is a condition that predominantly affects young women, and is associated with high-risk human papillomavirus infection (see below). In severe cases, there may be extensive involvement of the perineum, including the peri-anal area. The incidence of malignant change occurring in these lesions is low compared with that for the cervix. There is a tendency for intra-epithelial neoplasia to occur multifocally, with synchronous or metachronous involvement of vulva, vagina and cervix.

Squamous carcinoma

Squamous carcinoma (Fig. 19.3) is a tumour predominantly affecting elderly women in whom it is not usually associated with human papillomavirus infection. The recognition of associated intra-epithelial neoplasia may be difficult as it may occur in a so-called ‘differentiated’ form. The appearances are those of squamous carcinoma in any site; thus the tumour may be well, moderately or poorly differentiated. The prognosis is determined by the size, depth of invasion and degree of histological differentiation of the tumour, and the presence and extent of lymph node metastases, which predominantly affect the inguinal lymph nodes.

Fig. 19.3 Vulval squamous carcinoma. Surgical resection showing a large, fungating and invasive tumour on the vulva of an elderly patient.

In contrast to squamous carcinoma of the cervix, even minimally invasive disease in the vulva is associated with a risk of local lymph node metastasis, although this risk seems to be negligible for carcinoma invading to a depth of less than 1 mm. Tumour thickness greater than 5 mm and positive lymph nodes are associated with a poor prognosis.

Paget’s disease

The rare occurrence of mucin-containing adenocarcinoma cells within the squamous epithelium of the vulva is analogous to Paget’s disease of the breast (Ch. 18). Paget’s disease of the vulva tends to be chronic, with multiple recurrences. It may be indicative of an underlying invasive adenocarcinoma (in about 25% of cases), usually of skin adnexal origin, although, unlike the equivalent breast lesion, this is not usual. Adenocarcinomatous differentiation within the squamous epithelium has also been proposed as a possible explanation.

Other malignant tumours

Other malignant tumours of the vulva are rare. The most important of these are basal cell carcinoma, for which local excision is usually curative, and malignant melanoma which, as in other sites, generally has a poor prognosis.

Infections

Vaginal infections are common and often sexually transmitted. The organisms of most importance are: Gardnerella vaginalis, Neisseria gonorrhoeae, Candida albicans and Trichomonas vaginalis.

Vaginal adenosis

The occurrence of glands within the subepithelial connective tissue of the vagina is uncommon, and is believed to be due to a defect in embryological development. The lining of these glands is usually a mucinous cuboidal epithelium which may undergo squamous metaplasia. Vaginal adenosis particularly affected young females who were exposed to diethylstilbestrol in utero. This synthetic oestrogenic agent was used in the 1950s in the treatment of threatened miscarriage in the USA and, to a lesser extent, in the UK. Clear cell adenocarcinoma of the vagina may rarely complicate adenosis.

Vaginal intra-epithelial neoplasia

Vaginal intra-epithelial neoplasia is much less common than cervical intra-epithelial neoplasia but the same diagnostic criteria are applied. The lesion may co-exist with similar lesions of the vulva and cervix (reflecting the multicentric origin of squamous neoplasia).

Vaginal squamous carcinoma

Vaginal squamous carcinoma is an uncommon tumour predominantly occurring in older women. Pathologically, the tumour resembles squamous carcinoma of the cervix but it has a propensity to local invasion and radical surgery may be necessary.

Cervicitis

Non-specific acute and/or chronic inflammation is common in the cervix, particularly in the presence of an intra-uterine contraceptive device, ectopy (see below) or prolapse.

Chlamydiae are obligate intracellular organisms containing DNA and RNA, and are larger than viruses. Chlamydia trachomatis is a common sexually transmitted infection which is often recognised by its persistence following treatment for gonorrhoea in males (post-gonococcal urethritis). Chlamydiae can be isolated from the cervices of about 50% of asymptomatic female partners of these infected males and from women with chronic cervicitis. Chlamydial infection may produce subepithelial reactive lymphoid follicles, a condition sometimes given the label of ‘follicular cervicitis’.

Cervical polyps

Benign polyps of the cervix are common. They are composed of columnar mucus-secreting epithelium and oedematous stroma. Vessels may be prominent and there may be acute or chronic inflammation of varying severity. These polyps have no malignant potential.

Cervical microglandular hyperplasia

Cervical microglandular hyperplasia is a commonly seen complex glandular proliferation that may be confused with carcinoma. Small, tightly packed glands, lined by low columnar or cuboidal epithelium, may form polypoid projections into the endocervical canal. Accompanying acute inflammation and reserve cell hyperplasia (see below) are often seen. These changes may be seen in pregnancy and in users of the oral contraceptive pill, where they are the result of high levels of progestogen. Microglandular hyperplasia may also rarely be seen in post-menopausal women. It appears to have no malignant potential.

Aetiology

Squamous neoplasia of the cervix is associated with sexual activity; early age at first intercourse, frequency of intercourse and number of sexual partners are all risk factors. The sexual behaviour of the male partner is probably also of importance. There is probably no one single cause of cervical cancer or pre-cancer, but epidemiological evidence points to a sexually transmitted agent or agents. There is now compelling evidence that human papillomaviruses are implicated in the aetiology of cervical squamous neoplasia. Cigarette smoking is an independent risk factor; some contents of cigarette smoke, which can be detected in cervical mucus, may act as co-carcinogenic agents. The polycyclic aromatic hydrocarbons in cigarette smoke form damaging adducts with DNA; these have been demonstrated in cervical tissue at higher levels in current smokers.

Human papillomaviruses and neoplasia of the lower female genital tract

Genital warts or condylomata have been recognised for centuries. Only comparatively recently, however, has their viral aetiology been established. Electron microscopy showed the presence of viral particles, and immunohistochemistry (using antibodies to viral capsid antigen) and in situ hybridisation (using DNA probes) also confirmed their viral nature. Warts may affect the vulva but may also involve the cervix (Fig. 19.4). Moreover, it is now appreciated that human papillomaviruses (HPV) may infect the vulva, vagina and cervix in a non-condylomatous manner. Such infections show characteristic morphological features; most important of these is a specific cytoplasmic vacuolation called koilocytosis (Fig. 19.5). The features associated with human papillomavirus infection are:

• koilocytosis

• hyperkeratosis

• parakeratosis

• papillomatosis

• individual cell keratinisation (dyskeratosis)

• multinucleation.

Fig. 19.4 Florid condyloma of the cervix.

Fig. 19.5 Koilocytosis. Cytoplasmic vacuolation and pyknotic nuclei indicative of human papillomavirus infection of the cervix.

These morphological features are also common accompaniments of vulval, vaginal and cervical intra-epithelial neoplasia.

There are now more than 100 subtypes of human papillomavirus recognised and certain of these show a particular predilection for the lower female genital tract, notably HPV 6, 11, 16 and 18. HPV 6 and 11 are found in benign condylomata and are only rarely implicated in malignant transformation. HPV 16 and, to a lesser extent, 18 are found in cervical intra-epithelial neoplasia and in nearly 100% of cervical carcinomas. Other types, such as HPV 31 and 33, have also been reported in carcinoma. These are the oncogenic HPV types. Infection of the male genitalia by HPV is also seen; similar lesions to those seen on the cervix occur on the glans penis and prepuce, and may also be associated with neoplastic change.

Papillomavirus DNA may be present either extrachromosomally (episomal) or integrated into the host DNA. Integration of the viral genome into host DNA is usual in high-grade cervical intra-epithelial neoplasia (see below) and invasive cervical squamous carcinoma. The protein coding sequences of the viral early (E) or late (L) open reading frames appear to have a major role in oncogenesis. Most interestingly, the E6 protein of HPV type 16 is capable of binding to the cellular p53 protein to form a complex that neutralises the normal response of cervical epithelial cells to DNA damage (apoptosis mediated by p53), which may thereby allow the accumulation of genetic abnormalities. E6 protein of low-risk HPV types (e.g. 6 and 11) does not appear to form a complex with p53.

These events may explain why, unlike many other solid tumours, mutation of the p53 gene is an uncommon event in cervical carcinogenesis, as there is an alternative mechanism for its inactivation.

HPV 16 and 18 E7 proteins also have the ability to bind to the product of the retinoblastoma gene (RB1), thus affecting its tumour suppressor role.

HPV vaccination is now being implemented in many countries. The vaccine comprises virus-like particles produced by recombinant DNA technology.

Physiological and neoplastic changes in the cervical transformation zone

Before puberty, the squamo-columnar junction lies within the endocervical canal (Fig. 19.6). With the onset of puberty and in pregnancy, there is eversion of the columnar epithelium of the endocervix so that the squamo-columnar junction comes to lie beyond and on the vaginal aspect of the external os. This produces the clinical appearance of a cervical ‘erosion’, an unfortunate term, as the change is physiological. The term ectopy is more appropriate. The columnar epithelium is then exposed to the low pH of the vaginal mucus and undergoes squamous metaplasia. This is a physiological phenomenon, and takes place through the stages of reserve cell hyperplasia and immature squamous metaplasia. Reserve cells undermine the columnar mucus-secreting cells and multiply. This labile epithelium is called the transformation zone and is the predominant site for the development of cervical neoplasia.

Fig. 19.6 Epithelial changes in the cervical transformation zone.

Cervical intra-epithelial neoplasia (CIN) refers to the spectrum of epithelial changes that take place in squamous epithelium as the precursors of invasive squamous carcinoma. The severity of the lesion is assessed subjectively as grade (CIN) 1 (low grade), 2 or 3 (high grade), according to the level in the epithelium at which cytoplasmic maturation is taking place (Fig. 19.7). Abnormal nuclei are present throughout the thickness of the epithelium, and mitotic figures are not confined to the basal cell layer (Fig. 19.8). Any grade of cervical intra-epithelial neoplasia is potentially invasive, although the risk of invasion becomes greater as the severity of the lesion increases. The rate at which these intra-epithelial lesions progress and the proportion of cases that would progress if left untreated is uncertain, but probably about 11% of CIN 1 cases will progress to CIN 3 within 3 years. More than 12% of cases of CIN 3 would progress to invasion if left untreated; about 30% of cases would regress. The presence of abnormal mitotic figures is associated with progression. It is also the case that, in some young women, the lesions progress to invasive carcinoma more quickly (3 years or less). The categorisation of cervical neoplasia into low (CIN 1) and high (CIN 2 and 3) grade intra-epithelial neoplasia reflects the clinical management of the disease.

Fig. 19.7 Cervical intra-epithelial neoplasia (CIN) and invasive squamous carcinoma. Cervical intra-epithelial neoplasia. The concept of CIN refers to the level in the epithelium at which cytoplasmic maturation occurs. Grade 1 represents mild dysplasia; nuclear abnormalities throughout the epithelium, and cytoplasmic differentiation in the upper two-thirds are present. Grade 2 represents moderate dysplasia, with differentiation in the upper third of the epithelium. Grade 3 represents severe dysplasia and carcinoma in situ. Early stromal invasion. Invasion is < 1mm and there is a negligible risk of lymph node spread. Microinvasive carcinoma. Invasion is <3 mm in depth and the maximum horizontal dimension of the tumour is < 7 mm. There is still < 1% risk of lymph node spread. The presence of tumour within local lymphatic or vascular channels does not affect this definition. Occult invasive carcinoma. Invasion is > 500 mm³ and there is some risk of lymph node spread, but the tumour is still clinically undetectable.

Fig. 19.8 Cervical intra-epithelial neoplasia (CIN) grade 3. Note that there is minimal surface differentiation.

The terms low-grade and high-grade squamous intra-epithelial lesion (SIL) are used in some countries, notably the USA (Table 19.2).

Table 19.2Classification of HPV-associated intra-epithelial lesions of the cervix

Cytology screening programmes

Cytology screening programmes are sometimes referred to erroneously as cervical cancer screening. This is incorrect because the aim is to detect atypical cells by cervical cytology in the pre-invasive stage of the disease. The abnormal epithelium can then be eradicated by local measures, such as diathermy large loop excision of the transformation zone (LLETZ).

Cervical cytology is a simple, safe, non-invasive method of detecting precancerous changes in the cervix. The majority of specimens submitted to the pathology laboratory are taken from asymptomatic women as part of a national screening programme. The incidence of and mortality from invasive cervical cancer has fallen dramatically in communities where intensive screening has been carried out. The rates of cervical cancer would be at least 50% greater if there was no screening programme; attendance for regular screening prevents up to 90% of cervical cancer.

The examination of a cervical cytology specimen relies on the identification of abnormal (dyskaryotic) nuclei (Fig. 19.9). The degree of abnormality may be mild, moderate or severe, but does not always correlate with subsequent histological findings in a biopsy specimen. Therefore, even women with mildly dyskaryotic cervical cytology specimens should be referred for colposcopy (Table 19.3). Cytology is not a reliable means of detecting an invasive tumour; the diagnosis of invasive carcinoma of the cervix is largely clinical and is confirmed by biopsy of suspicious areas of the cervix. The morphological abnormalities of the nucleus (dyskaryosis) in cervical cytology specimens are:

Fig. 19.9 Cervical cytology. Normal. Herpes virus infection; note the large multinucleated cell. Koilocytosis; the sharply defined perinuclear pallor indicates HPV infection. Dyskaryosis; enlarged nuclei with a coarse chromatin pattern. Papanicolaou staining; hence ‘Pap’ test.

Table 19.3 Clinical consequences of cervical cytology

In many centres liquid-based cytology (LBC), in which cells are received by the laboratory suspended in fluid and prepared as a near monolayer on a slide and stained, has replaced the smearing of cells onto a glass slide from a spatula.