The labia majora and minora of the vulva are covered by keratinising squamous epithelium (Fig. 21.2) which undergoes very little hormonal change during the menstrual cycle. The outer surfaces of the labia majora are hair-bearing. The inner surfaces have many sebaceous glands and apocrine sweat glands, the secretions of which provide protection against infection and local damage to the skin.

Fig. 21.2 Vulval skin showing stratified squamous keratinising epithelium with skin appendages (H&E).

The labia show pigmentation from the age of puberty, diminishing on the inner aspect of the labia minora where only a thin layer of keratin is present. This epithelium extends to cover the vestibule as far as the hymen. The vagina and the urethra open onto the vestibule of the vulva. Mucin secreting glands are present on either side of the vaginal introitus, including Bartholin’s glands, which are situated in the lower vaginal wall, providing protection and lubrication.

Developmentally, the upper two-thirds of the vagina is formed by fusion of the two mullerian ducts in utero. The tube thus formed differentiates into the uterus and cervix above and unites distally with the urogenital sinus to form the vagina. Thus the lower third of the vagina has a different embryological origin from the upper two-thirds. The importance of this dual derivation lies in the fact that the mullerian epithelium in the upper two-thirds is initially columnar in type, but undergoes metaplasia in utero to squamous mucosa on exposure to the acid pH of the vagina. Normally the change to squamous epithelium is complete, but the process may be interrupted, leading to persistence of glandular tissue in the adult vagina, a condition known as vaginal adenosis (see Fig. 21.99).

Under normal conditions, the vagina is lined by stratified squamous non-keratinising epithelium throughout (Fig. 21.3), usually showing no hair follicles, sweat glands or sebaceous glands to weaken its surface. The mucosa is subject to cyclical changes under the influence of the sex hormones.

Fig. 21.3 Vaginal squamous mucosa is not normally keratinised and is hormone sensitive, in contrast to vulval skin. The squamous cell cytoplasm is clear in this section due to the presence of glycogen (H&E).

The cervix is a cylindrical fibromuscular structure of variable length, within which lies the endocervical canal connecting the body of the uterus at the internal os with the vagina at the external os. The lower portion of the cervix protrudes into the vagina, forming the anterior, posterior and two lateral fornices in the upper vagina where pooling of secretions and exfoliated cells occurs. The outer aspect of the cervix, known as the ectocervix or portio vaginalis, is covered by squamous mucosa in continuity with vaginal epithelium distally and with the lining of the endocervix at or near the external os (Fig. 21.4).

Fig. 21.4 Section of adult cervix showing the ectocervix covered by non-keratinising squamous epithelium, the external os and the endocervical canal running towards the body of the uterus. Endocervical crypts face downwards and are lined by a single layer of columnar epithelial cells (H&E).

The endocervical canal is not exposed to the vaginal pH and therefore retains its glandular lining of tall columnar epithelium, with an inconspicuous layer of reserve cells beneath. resting on basement membrane. The glandular mucosa forms branching crypts that extend into the stroma of the cervix in a racemose pattern for a distance of up to 5 mm; the orifices facing distally. The canal itself is narrow, being only a few millimetres wide, and in health, is filled by a plug of mucus. During the menstrual cycle the physical properties of the cervical mucus change. Prior to ovulation the mucus is dilute, and when spread on to a slide and dried, it forms a fern-like pattern (Fig. 21.5). After ovulation, the mucus becomes thicker and no longer demonstrates ferning.

Fig. 21.5 Cervical mucus in a conventional/direct cervical smear taken at midcycle showing a ferning pattern due to the effects of oestrogen.

It will be apparent from the above description that there is a point of junction between the squamous epithelium of the ectocervix and the lining of the endocervical canal. This meeting point is referred to as the squamocolumnar junction. The changes that occur in this area are of crucial significance in cervical pathology.

Squamocolumnar junction and transformation zone

Major changes in the size and shape of the cervix take place during reproductive life. Before puberty, the squamocolumnar junction forms the outer boundary of the endocervical glands, known as the ‘original’ squamocolumnar junction, coinciding with the site of the external os.

After puberty, the location of the squamocolumnar junction changes as the cervix alters in shape. The endocervical mucosa of the lower canal, including the underlying crypts, is everted and comes to lie on the ectocervical aspect of the cervix, resulting in an ectropion or ectopy (Fig. 21.6). An ectropion is clinically visible as a reddened zone extending out from the external os. It is sometimes mistakenly called an erosion, although no actual ulceration is present and the condition is physiological.

Fig. 21.6 Ectropion. At menarche and during certain phases of reproductive life, the squamocolumnar junction is everted so that the endocervical glands come to lie on the outer aspect of the cervix, producing an ectropion (H&E).

Endocervical eversion is followed by progressive metaplasia of the exposed mucosa to more protective squamous epithelium under the influence of the vaginal pH, the term metaplasia referring to a change from one type of epithelium to another. The metaplastic process arises in the reserve cell population that normally replenishes the columnar epithelium. Instead, these reserve cells differentiate as immature squamous metaplastic cells beneath the single layer of columnar cells on the surface (Fig. 21.7A).

Fig. 21.7 (A) Reserve cell hyperplasia is seen beneath everted columnar epithelium. The cells are starting to show multilayered immature squamous metaplasia, forming a transformation zone. (H&E). (B) The transformation zone (between arrows) is now maturing but its site of origin is marked by the position of the first endocervical gland. Note the plug of mucus covering the surface (H&E).

The metaplastic layers increase and mature progressively to form a new normal squamous mucosa indistinguishable from the original squamous epithelium of the ectocervix. The underlying endocervical crypts remain facing on to the ectocervix as a permanent marker of the site of the original squamocolumnar junction and this can be recognised colposcopically as well as in histological section (Fig. 21.7B). Glands with orifices obstructed by the metaplastic process are prone to distend with mucus, even becoming visible macroscopically as rounded nodules on the ectocervix, known as nabothian follicles.

The extent to which eversion of the endocervix occurs varies in different women and the new junction may be asymmetrical in relation to the external os, with ectropion covering one or both sides of the ectocervix, even extending on to the vaginal walls. Ectropion is liable to recur with certain hormonal events. The most obvious of these is pregnancy, during which ectopy is a common finding due to enlargement of the cervix under the influence of progesterone. Some types of oral contraceptive therapy have a similar effect. After the menopause the cervix shrinks and the squamocolumnar junction migrates into the endocervical canal.

The area of metaplastic epithelium proximal to the original squamocolumnar junction is referred to as the transformation zone since it is an area of epithelial instability. Immature metaplastic cells appear to carry an extra risk of neoplastic change.

Structure of stratified squamous epithelium (Figs 21.8, 21.9)

The germinal layer is composed of a single row of small regular cells adhering to a basement membrane and showing signs of active growth. These undifferentiated cells are referred to as the basal cells. Above this layer it is possible to distinguish parabasal cells, immature and crowded, lying two to three cells deep. These cells mature into an intermediate layer of variable thickness in which the cells have more cytoplasm, the nuclei still show a recognisable chromatin pattern and the cells are bound to each other by intercellular cytoplasmic bridges.

Fig. 21.8 Stratified squamous mucosa from the cervix during reproductive life shows a distinct row of primitive basal cells resting on a thin basement membrane. Above this there is progressive maturation from 3 to 4 parabasal cell layers to an intermediate cell layer with more cytoplasm and preserved nuclear structure, while the uppermost superficial cells have condensed pyknotic nuclei and voluminous cytoplasm (H&E).

Fig. 21.9 Postmenopausal atrophy of ectocervical squamous mucosa. The epithelium is thinned, with crowding of squamous cells due to their reduced cytoplasm. The basal layer is still distinct and polarisation of cells is visible (H&E).

In fully mature cervical squamous mucosa there is a superficial layer consisting of cells that do not normally mature any further. Intercellular bridges are not highly developed at this level so that the cell bonds are weaker. Superficial cells are actually dead or dying and exfoliate spontaneously. Sometimes a thin upper layer of cells with dark cytoplasmic keratohyaline granules may be present in histological sections, formed of the cells that precede keratinisation, but a keratinised layer is not seen under normal conditions.

Mucosal thickness depends upon hormonal status as the parabasal, intermediate and superficial layers are all hormonally responsive. Under the influence of oestrogen a superficial layer develops in about 4 days. This multilayered epithelium provides a barrier against external injuries and stores nutrients in the form of glycogen.

These small primitive cells are parent to all the cells in the squamous mucosa that migrate to the surface, die and exfoliate. They are difficult to recognise in a smear with confidence and are likely to be sampled only rarely due to their deep position in the mucosa and their firm attachment to the basement membrane. They are said to occur in short rows of small regular cells with sparse green cytoplasm, oval nuclei and a high nuclear/cytoplasmic (N:C) ratio. The chromatin pattern is fine and several chromocentres may be present.²

Parabasal cells (Fig. 21.10)

Fig. 21.10 A row of parabasal cells, rounded or polygonal in shape, dissociated and in small groups. The cytoplasm is dense and cyanophilic, the nuclei stain darkly and the nuclear/cytoplasmic ratio is high compared with the intermediate cells at lower edge.

Parabasal cells may predominate in atrophic smears from postmenopausal women. In younger women postnatal atrophy or high dose progesterone oral contraception produce similar changes. With poor fixation and in some inflammatory states parabasal cytoplasm may be amphophilic, taking up eosin staining centrally with peripheral cyanophilia.

Intermediate cells (Fig. 21.11)

Fig. 21.11 Intermediate and superficial squamous cells. The former have translucent green cytoplasm with a tendency to fold at the periphery; the nuclei show a vesicular chromatin pattern and the nuclear/cytoplasmic ratio is low. The superficial cells are large, flat, polygonal and dissociated, with pinkish orange cytoplasm. The tiny dark nuclei have lost their chromatin pattern.

The cells lie in tight groups or discretely in the smear, depending upon the hormonal state. In the second half of the cycle, when cell clumping is greatest, the cytoplasm becomes ragged and may disintegrate altogether, leaving bare nuclei. Under the influence of high levels of progesterone, intermediate cells tend to accumulate glycogen, forming an irregular central deposit of pale yellow stained material.

Superficial cells (Fig. 21.11)

Cells from the granular layer, if formed, display small dark blue keratohyaline granules evenly distributed in the cytoplasm. Nests and aggregates of benign squamous cells known as epithelial pearls and rafts are sometimes seen in normal smears (Figs 21.12, 21.13).

Fig. 21.12 An epithelial spike/raft of parakeratotic squamous cells.

Fig. 21.13 Concentric epithelial pearls can be seen in normal cervical cytology samples.

Fig. 21.14 Vulval contamination of a cervical smear is suggested by these yellowish orange anucleate squames. They are also seen in direct vulval scrapes.

Anucleate squames are often present in combination with granular cells, indicating completion of keratinisation. The cells are a normal finding in vulval scrapes. Numerous anucleate squames in a smear from the cervix suggests an area of keratinisation (hyperkeratosis) due to irritation from, for instance, prolapse or the wearing of a pessary. Hyperkeratosis also occurs in human papillomavirus infection, leukoplakia and in some cases of cervical carcinoma. The presence of anucleate squames is, however, of low predictive value for these conditions. Less mature cells from the deeper layers may also become anucleate, usually in association with degenerative changes.

Endocervical cells (Fig. 21.15)

Fig. 21.15 Endocervical cells in sheets and groups, their columnar form giving a picket-fence (arrow) or honeycomb appearance (arrowhead). The cytoplasm is delicate and nuclei are rounded with a vesicular chromatin pattern.

Fig. 21.16 Bare nuclei are frequently seen around endocervical cell groups. The nuclei become swollen and flattened like a fried egg when unsupported by cytoplasm, with pale but uniform chromatin. These nuclei may be of reserve cell origin.

Sometimes cilia are visible, staining pink with eosin, attached to the terminal bar at the luminal pole of the cell (Fig. 21.17). Cilia are more commonly seen postmenopausally but may also arise with tuboendometrioid metaplasia which can occur in the process of healing of the cervical mucosa after injury such as post-conisation.

Fig. 21.17 A few ciliated endocervical cells form part of the normal glandular epithelium of the cervix but the cilia are not often seen as they are prone to undergo degenerative changes.

Occasionally endocervical cell cytoplasm may become distended by mucus producing a goblet cell appearance. Care should always be taken to examine the overall morphology of these cell groups to exclude the rare possibility of intestinal-type glandular intraepithelial neoplasia (see Ch. 24) (Fig. 21.18) Background mucus from endocervical cells stains variably, either faintly green or tinged with pink. The amount of mucus present in a sample, its quality and distribution also vary considerably (Fig. 21.19). With conventional smears a ‘ferning’ effect may be evident at the time of ovulation (Fig. 21.20). This pattern is not seen with liquid-based cytology preparations.

Fig. 21.18 Endocervical cells with goblet cell morphology due to distension with mucin.

Fig. 21.19 (A) Background mucus stained pale pink or green is a useful indicator of sampling from the cervix. Vaginal and vulval smears usually contain little or no mucus. (× MP) (B) The mucous plug of the cervix may be spread on a smear largely intact. The plug contains inflammatory cells but the remainder of the smear will be clean, in contrast to a genuine infection (× LP).

Fig. 21.20 Cervical mucus spread in a cervical smear taken at midcycle showing a ferning pattern due to the effects of oestrogen (× MP).

Endocervical cells may show considerable variation in nuclear size within a group, although the polarity is maintained (Fig. 21.21).

Fig. 21.21 Note the variation in nuclear size in occasional endocervical cells (most prominent at the bottom), but polarity is maintained.

These cells are a normal constituent of smears from the cervical os once the transformation zone has developed (Fig. 21.22). While immature, they do not exfoliate spontaneously but can be lifted from the surface of the cervix by the abrading action of a spatula or brush. With progressive maturation, the cells increasingly resemble intermediate and superficial cells from the original ectocervix and therefore cannot be recognised as a separate cell population in cervical samples.

Fig. 21.22 A metaplastic group showing angular and polygonal cells with dense blue-green cytoplasm and nuclei resembling those of endocervical cells.

The cytoplasmic projections that give a spidery contour to the single cells result from their forcible removal during sample taking. In metaplastic cell sheets the cytoplasmic projections may appear as fine intercellular bridges (Fig. 21.23). In the unstable environment of the transformation zone, premature keratinisation of these cells may occur and the cytoplasm is then a deep orange colour. Other degenerative changes such as vacuolation and the presence of intracytoplasmic polymorphs may be seen, even in the absence of significant inflammation. Degenerative nuclear changes may also occur if maturation of the transformation zone is arrested; these include pyknosis, referring to condensation of the entire nucleus, and fragmentation or dissolution of chromatin, referred to as karyorrhexis and karyolysis, respectively.

Fig. 21.23 Immature squamous metaplasia showing spidery cytoplasmic projections due to forceful removal of the cells from the mucosa.

Endometrial cells may be seen normally in cervical samples up to 12 days from the onset of menstruation. Factors influencing their presence beyond this may reflect underlying endometrial pathology in a woman over 40 years of age or could be due to exogenous hormonal manipulation such as hormone replacement therapy or oral contraceptive use; tamoxifen use, intrauterine device carriage and dysfunctional bleeding are further potential sources of endometrial cells outside the allowed phase of the cycle.

The appearance of endometrial cells varies with the stage of the cycle and their degree of preservation. During and shortly after menstruation they are grouped in well-formed tight three-dimensional clusters with a peripheral rim of epithelial cells and a central core of stromal cells (Fig. 21.24). Degenerative changes quickly supervene, with crumpling of the nuclei and disorganisation of the cells. These then stand out as small clusters of densely hyperchromatic crowded cells (Fig. 21.25), in contrast to the larger, more regular and better-preserved groups of endocervical cells. Neutrophil polymorphs are often seen within endometrial clusters. Endometrial cells of both epithelial and stromal origin can be very well preserved in liquid-based preparations reflecting prompt fixation at the time of sample taking. Loose aggregates and single histiocytes are often associated with shed endometrial cells and may be confused with severely dyskaryotic cells if their reniform nuclei and delicate cytoplasm are overlooked.

Fig. 21.24 Endometrial cells in a well-preserved group showing a central core of compact stroma surrounded by a looser regimented layer of small dark epithelial cells. This is the characteristic appearance of endometrial cells shed early in menstruation.

Fig. 21.25 Degenerate endometrial cells shed towards the end of menstruation are darkly stained and maybe associated with histiocytes and debris. Cell detail can be difficult to make out at this stage and awareness of the timing of smear collection in the cycle is important.

The cellular changes in endometrial cells in cervical smears in pathological states, such as endometrial hyperplasia or neoplasia, are described in Chapter 26.

Subcolumnar reserve cells are infrequently identified in cervical smears as they do not exfoliate. Possibly they are represented by some of the bare nuclei seen in the vicinity of some endocervical groups. When reactive reserve cell hyperplasia has occurred the cells can sometimes be identified as syncytial groups of small crowded cells with indistinct cell borders and round darkly stained nuclei which often overlap (Fig. 21.26). They may be distinguished from the cell groups of glandular dyskaryosis by the lack of architectural abnormalities, the smaller size and uniform chromatin pattern of their nuclei and by the presence of associated bare nuclei and normal endocervical cells in conventional smears.

Fig. 21.26 Reserve cell hyperplasia in a cervical smear. This crowded group of glandular cells includes a population of smaller darker nuclei, presumed to represent reserve cells.

These are the commonest of the non-epithelial cells found in normal smears, their presence being physiological within the mucous plug of the cervix. They can be found in large numbers without necessarily implying significant infection, although they are usually increased in cases of cervicitis or vaginitis and also in established malignant disease of the cervix. The clinical context and the overall cytological findings are important in evaluating their significance. There may be problems in the interpretation of a sample if the epithelial cells are largely obscured by polymorphs. This problem is overcome to a considerable extent by the use of liquid-based methods of preparation (see Fig. 21.57).

Macrophages are sometimes seen as part of the inflammatory cell population, especially following menstruation and in postmenopausal women. They are extremely variable in size and appearance, but can generally be distinguished from parabasal or columnar cells by their ill-defined foamy cytoplasm, their eccentric bean-shaped nuclei, and the presence of ingested particulate material in some instances. However, many of them have small round central nuclei and do not contain phagocytosed particles, making identification less certain. It is helpful when in doubt to examine neighbouring cells as these frequently include other macrophages with more typical features (Fig. 21.27).

Fig. 21.27 Macrophages are dissociated cells, varying greatly in size and appearance but often small in cervical smears. This collection includes several with bean-shaped nuclei and foamy cytoplasm, features typical of macrophages.

Macrophages, although usually dissociated cells, may be loosely aggregated especially in postmenstrual smears. They may become multinucleated and very large, forming giant cells, a phenomenon most often seen in postmenopausal women (Fig. 21.28).

Fig. 21.28 Multinucleated macrophages or giant cells are a non-specific finding, especially after the menopause. They are also seen in granulomatous inflammation or repair and after radiotherapy.

These cells are usually scanty, forming a minor component of the inflammatory cell population in some normal women. They are present in larger numbers in follicular cervicitis, in association with tingible-body macrophages (see Fig. 21.67).

Eosinophils, basophils (mast cells) and plasma cells (see Fig. 21.58) are occasionally seen in samples, recognisable by their characteristic morphology.

Spermatozoa are seen in postcoital smears, even several days after intercourse (Fig. 21.29).

Fig. 21.29 Spermatozoa are a common finding in cervical samples. They show darkly stained ovoid sperm heads with some preserved tails.

Cytological specimens can be contaminated at any stage in the collection, transmission or laboratory preparation of the sample. Liquid-based cytology preparations are less prone to contamination from these sources. In addition, cervical samples may include extraneous material from the vagina or vulva, even including parasites or their ova from the digestive tract, especially in those parts of the world where parasitic infestations are common.³ Even outside such endemic areas parasites are encountered from time to time in cervical smears and their identification is important in patient management.

Ova of the threadworm Enterobius vermicularis⁴ (Fig. 21.30) are not infrequently seen in smears from infected patients, especially if there is poor personal hygiene. The eggs are oval and are smaller than schistosome ova, with a smooth double-walled shell, often with one side flipped over. The larva can usually be recognised within. Occasionally adult forms can be identified (Fig. 21.31).

Fig. 21.30 Ova of Enterobius vermicularis, showing the thick glassy eosinophilic capsule and larva within.

Fig. 21.31 Adult Enterobius vermicularis worm in a conventional cervical smear.

Descriptions of Ascaris lumbricoides, Taenia coli,⁵ Trichuris trichura, Hymenolepis nana⁶ and the microfilaria of Wuchereria bancrofti^7,8 have been recorded. Schistosoma haematobium, S. japonicum and S. mansoni ova can be identified in smears as elliptical ova, larger than those of the threadworm E. vermicularis. The first two are the common types of schistosome ova found in cervical smears and are distinguished by the presence of either a terminal or lateral spine, respectively (Fig. 21.32).⁹ The trophozoites of Balantidium coli may be seen in patients with intestinal infestation by this uncommon protozoal organism (Fig. 21.33).

Fig. 21.32 (A) Schistosoma haematobium ovum in a conventional/direct cervical smear taken from a patient with vulval schistosomiasis. Note the size of the ovum compared with the squamous cells, and the terminal spine. (B) Vulval biopsy from the same patient showing numerous ova within the dermis and a surrounding foreign body reaction (H&E).

Fig. 21.33 Balantidium coli, a protozoal organism pathogenic in the large bowel, is a rare finding in smears and may not signify infection. In this asymptomatic patient only occasional trophozoites were seen. No treatment was given.

Pediculus humanus, the body louse, and the pubic louse, Phthirus pubis, are seen occasionally in cervical smears (Fig. 21.34). The louse may be damaged during smear preparation, with fragmentation of the tail part from head and legs.

Fig. 21.34 Pubic (crab) louse found in a cervical smear.

Many external contaminants have been described, including pollen and insects due to atmospheric contamination, and also various fungi, either from contaminated laboratory solutions, the water supply or from the atmosphere (Fig. 21.35). Particulate material from sources such as tampons or glove powder is usually easily identified (Fig. 21.36).¹⁰

Fig. 21.35 (A) Contamination of a cervical sample by fungal spores and hyphae, probably from the atmosphere. (B) A plant particle (scleroid) seen occasionally in smears due to atmospheric contamination.

Fig. 21.36 (A) Cotton fibres in a conventional/direct cervical smear. (B) Glove powder can often be seen in conventional/direct smears. Even without polarised light, the characteristic Maltese cross structure can be seen at the centre of some of the starch particles.

A refractile brown deposit overlying the central portion of the cytoplasm, known as ‘cornflake artefact’, may be seen in samples – particularly on direct smears and may obscure nuclear detail (Fig. 21.37). This is thought to result from the trapping of air on the surface of cells during mounting, especially in thickly spread direct smears. Inadequate removal of spray fixative containing Carbowax may cause similar problems. The artefact may be so marked as to require a further sample for accurate assessment.

Fig. 21.37 ‘Cornflake’ artifact refers to a brown slightly refractile deposit overlying the nuclei, sometimes obscuring nuclear detail completely.

Lubricant contamination may occasionally be seen with some liquid-based cytology preparations. The morphological appearances are variable and include amorphous blue deposits and stringy eosinophilic background material.¹¹

Carryover of cellular material from one sample to another may occur during sample preparation, including staining, posing difficulty in assessing the findings. The contaminant cells are often distributed along the upper or side edge of the slide in direct smears and may be in a slightly different plane of focus from the rest of the cells. If there is any abnormality in the carryover it is confined to cells at this site and does not appear to relate to the morphology of other cells present.

Liquid-based thin layer cytological preparations (LBC), usually collected by brush sampling, reduce the rate of obscured and inadequate samples by removing much of the inflammatory debris and blood and providing a representative sample of all of the material from the spatula, most of which is usually discarded in routine smear preparation. Since 2008, the entire UK NHSCSP has adopted LBC preparations. The Bethesda System in America has adopted 5000 cells per sample as a minimum threshold for adequacy.¹³ With its 3–5 yearly routine screening interval, the UK NHSCSP’s more stringent criteria are considered more appropriate than those adopted by TBS, but none have yet been agreed nationally, and sample adequacy is the subject of a Health Technology Assessments (HTA) study which has yet to report (see Ch. 22).

Oestrogens promote growth and maturation of stratified squamous epithelium up to and including the superficial layer. Smears contain many superficial epithelial cells when the oestrogen level is high and unopposed by progesterone, as in the first half of the menstrual cycle. These cells lie quite flat and are generally discrete; the smear background is noticeably free of polymorphs (Fig. 21.41B).

A small dose of oestrogen can cause atrophic squamous epithelium to develop into epithelium with several layers of intermediate cells. When the dose of oestrogen is increased or prolonged, intermediate cells mature into superficial cells.

Fig. 21.42 Oestrogenic effects are manifested by the appearance of large, flat dissociated eosinophilic superficial squamous cells in a clean background.

Once the stratified squamous epithelium has matured under the influence of oestrogen, progesterone causes rapid desquamation of the topmost layers. The intermediate cells develop curled edges giving a folded appearance, and the cytoplasm often contains glycogen, staining yellow at the centre of the cell. Exfoliation occurs in compact clusters of cells in which the margins of individual cells are indistinct.

Many Döderlein’s bacilli (lactobacilli) and leucocytes appear in the smear. They bring about cytolysis of the intermediate cells, causing dissolution of the cell cytoplasm (Fig. 21.43). As a result, numerous naked vesicular nuclei are present and the smear may appear hypocellular. Further progesterone induces the appearance of boat-shaped navicular cells (Fig. 21.44).

Fig. 21.43 Progesterone effects include maturation of squamous cells to intermediate level, with increased glycogen content; proliferation of lactobacilli and cytolysis follow further exposure to progesterone.

Fig. 21.44 Navicular cells containing yellow stained glycogen are characteristically boat-shaped with the nucleus pushed to the periphery of the cell and the cytoplasm folded at the cell margins.

When there is mild oestrogen deficiency, as may be encountered at the time of the menopause, the cytological pattern is difficult to distinguish from that of progesterone or androgen stimulation. The clusters are, however, usually slightly smaller, often containing no more than 10 cells.

If progesterone is administered to patients with an atrophic mucosa, maturation of the squamous epithelium including the superficial layers is the initial result. Administration of progesterone to patients with pre-existing mature epithelium leads to disappearance of the superficial layer and no superficial cells are found in the smear.

Administration of this hormone when the stratified squamous epithelium is atrophic and smears are composed exclusively of parabasal cells results in a predominance of intermediate cells (Fig. 21.45). The smears are particularly rich in cells.²⁵ If androgens are administered to patients with fully mature stratified squamous epithelium, the opposite effect is seen: superficial cells disappear, to be replaced by intermediate cells. Prolonged administration of androgens produces a smear pattern with cytolysis.

Fig. 21.45 Androgenic effects include maturation of an atrophic smear to intermediate cell level. The background is usually clean, lacking the cytolysis seen with progesterone activity.

These are best revealed in vaginal smears taken from the lateral vaginal wall, preferably sequentially; but the changes are mirrored by the findings in cervical smears. Vulval tissues are much less responsive to cyclical hormonal fluctuations, although subject to hormonally determined development and atrophy at the menarche and the menopause, respectively.

Sexual maturity

When menstrual cycles become ovulatory, the following consecutive patterns can be expected (see Fig. 21.46):

• Menstrual phase (1st–5th day). The smear contains erythrocytes, leucocytes, endometrial cells, and superficial and intermediate epithelial cells

• Proliferative or follicular phase (6th–10th day). First a large number of histiocytes appear, often in loose aggregates. These are mainly derived from endometrial stromal cells shed in a process known as ‘the exodus’. There are also many early intermediate squamous epithelial cells lacking the folding seen under the influence of progesterone. Polymorphs and endometrial cells are present, the latter decreasing in number up to the 10th–12th day. Towards the end of the proliferative phase superficial squamous cells increase in number

• Ovulation (11th–13th day). During this phase there are numerous superficial epithelial cells lying flat and obviously discrete. The pattern is ‘clean’, that is, practically without leucocytes and with very few bacteria. In some conventional smears the endocervical mucus creates a pattern of ferning on the slide (see Fig. 21.20)

• Secretory or luteal phase (14th–21st day). The pattern changes abruptly after ovulation: now intermediate squamous cells predominate. Within several days the characteristic progesterone pattern appears, with folding and clustering of intermediate cells, the reappearance of polymorphs and many lactobacilli

• Towards the end of the cycle, marked cytolysis develops, accompanied by a further increase in polymorphonuclear leucocytes. Just before menstruation, the proportion of mature epithelial cells increases once again. A few women also shed endometrial cells several days before menstruation. In some women with normal ovulatory cycles, the characteristic progesterone pattern is absent and only the midcycle peak of oestrogenic effects points to ovulation.

Fig. 21.46 These three fields show some of the characteristic features of cervical samples at different stages of the menstrual cycle. (A) Menstrual phase with endometrial cells. (B) Early proliferative phase with stromal histiocytes (‘exodus’). (c) Late secretory phase with marked cytolysis.

Pregnancy

In the event of pregnancy, the corpus luteum does not involute but instead grows larger, producing increasing amounts of progesterone and oestrogen. The cytological pattern of pregnancy is that of heightened progesterone activity, with clustering and folding of intermediate cells and the presence of navicular cells. These boat-shaped cells are distended with yellow glycogen, having a rim of folded cytoplasm and the nucleus pushed to the periphery (Fig. 21.44). The changes are most pronounced in the third trimester, when numerous lactobacilli are seen and the accompanying cytolysis is at its height.

About 3 months after conception, the placenta takes over the task of producing progesterone and oestrogens from the corpus luteum. Arias-Stella changes, seen in the endometrium in some pregnancies, may also develop in endocervical glands and have been described as a rare finding in cervical or vaginal smears.^26,27 The cells have an exaggerated secretory pattern as seen in the endometrial glands, and show enlarged degenerate hyperchromatic nuclei with intranuclear inclusions. The association with pregnancy and lack of any preserved abnormal chromatin pattern, combined with awareness of this entity, should ensure correct assessment of the findings.

Syncytiotrophoblastic cells are multinucleated cells from the outer surface of the chorionic villi and hence are foetal in origin, occurring only rarely in cervical cytology samples, for instance from patients with placenta praevia or in the presence of a threatened miscarriage. The cells are large, with an average of 50 nuclei per cell and a characteristic coarse-grained chromatin pattern resembling coarsely ground pepper. The number of nuclei is large in relation to the amount of cytoplasm. The cytoplasm is granular, in contrast to that of a histiocytic giant cell.

Cytotrophoblastic cells also cover the villi. They are cuboidal cells with central nuclei. Cytologically, these cells cannot be firmly identified and may mimic neoplastic cells due to their prominent nucleoli, coarse chromatin pattern and high N:C ratio (Fig. 21.47).

Fig. 21.47 Trophoblastic cells are difficult to identify with confidence. This group of degenerate cuboidal cells with enlarged hyperchromatic nuclei is presumed to be of cytotrophoblastic origin. The cells were present in a smear taken 2 weeks after a miscarriage. Follow-up samples have been normal.

Intact placental villi have occasionally been identified in samples taken postpartum²⁸ As illustrated in Figure 21.48, a villus may be recognised by its size and form, with degenerate trophoblastic cells and inflammatory debris coating a long three-dimensional structure which has a translucent quality internally.

Fig. 21.48 (A) Placental villus in a conventional/direct cervical smear taken 6 weeks after delivery. The structure appears three-dimensional on low power. (B) High-power magnification shows degenerate trophoblasts on the outer surface with a few adherent inflammatory cells.

Decidual cells are modified endometrial stromal cells responding to the high circulating progesterone levels in pregnancy or to high progesterone content oral contraceptives. Decidual changes may also occur in the stromal cells of the cervix and are occasionally sampled in smears taken during or shortly after pregnancy. The cells are swollen and pale due to their content of glycogen. They have abundant clear, sometimes vacuolated cytoplasm and central large nuclei. The chromatin is often smudged and ill-defined and nucleoli are prominent. Decidual cells can be recognised by the fact that, in contrast to squamous epithelial cells, they are not flat but convex; the cells are round to oval and the cytoplasm appears to be less dense than that of epithelial cells (Fig. 21.49).²⁹

Fig. 21.49 (A,B) Decidual cells seen in a postnatal cervical sample taken at the same time as the accompanying biopsy. In the sample the cells have dense rather convex cytoplasm with bland nuclei, as in the biopsy (H&E).

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