NORMAL ANATOMY AND HISTOLOGY OF THE FEMALE GENITAL TRACT
Female genital tract is composed of:
- Vulva
- Vagina
- Cervix
- Uterus
- Fallopian tube
- Ovaries.
Figure 1.1 shows the different parts of the female genital tract.
Vulva
This is the external female genital part which is composed of mons pubis, labia majora, labia minora, clitoris, prepuce and vestibule.
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Mons pubis: The soft fibrofatty tissue over the pubic bone and symphysis pubis is known as mons pubis.
Labia majora: These are laterally situated two soft folds of skin that extend from the mons pubis to the perineum.
Labia minora: They are situated medial to labia majora. Anteriorly the two ends of labia minora encircle the clitoris.
Clitoris: This is the soft erectile tissue that represents male counterpart of penis.
Vagina
This is 8 to 9 cm fibromuscular tube that joins the vulva with the uterus. The upper part of the vagina merges with ectocervix. There is a free space between the cervix and the posterior vaginal wall which is known as posterior vaginal fornix. In this potential space the exfoliated cells accumulate.
Histology
It has three layers:
- The inner mucosal layer is composed of nonkeratinizing stratified squamous epithelium.
- The middle muscular layer.
- Outer connective tissue layer. The squamous epithelial layers of the vagina is responsive to estrogen and contains glycogen. This glycogen provides nutrient to the bacterial flora of the lower genital tract. The lactic acid produced by the bacterial flora maintains acidic pH and protects from pathogenic invasion.
Uterus
The uterus consists of three parts: fundus, body, and cervix.
Fundus: This is the uppermost part of the uterus and it is situated above the origin of the fallopian tube.
Body: This is the central wide part of uterus.
Histology of Body and Fundus
Uterus consists of (1) inner endometrium, (2) middle myometrium, and (3) outer serosa.
The mucosal lining of endometrium is composed of columnar cells. The endometrium undergoes cyclical changes and shredded out in regular interval if pregnancy does not occur. There are two layers of endometrium:
- Outer functionalis layer: This is superficial layer and is regularly shredded out during menstruation.
Cervix
The cervix is a cylindrical tube of 3 cm in length in nulliparous woman. This is the lower portion of the uterus and is connected with the body of uterus by isthmus. The upper part of the cervical canal is connected with uterus by internal os and the lower part of it opens into the vagina through an orifice known as external os. The part of the cervix that remains within the vagina is known as ectocervix (Figure 1.2). The endocervical canal is the passage in between the external and internal os of the cervix.
Histology of Cervix
Ectocervix: It is lined by non-keratinizing stratified squamous epithelium. It consists of three layers (Figure 1.3): 1) Basal or parabasal zone is composed of basal and parabasal cells, 2) Intermediate zone is made of intermediate squamous cells, and 3) Superficial zone is formed by flattened superficial squamous cells.
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Figure 1.4A: The section shows tall columnar lining and also subepithelial endocervical glands (Hematoxylin and eosin stain)
Figure 1.4B: Endocervical columnar lining epithelial cells in higher magnification (Hematoxylin and eosin stain)
Endocervix: It is made of single layer of mucus secreting tall columnar cells with basally placed nuclei (Figure 1.4 a, b). The endocervical lining often gives picket fence appearance. The same columnar cell also makes the lining of the endocervical glands.
Transformation Zone (TZ)
This is also called as squamocolumnar junction. It is the meeting point between the stratified squamous epithelium of the ectocervix and mucin secreting columnar epithelium of the endocervix (Figure 1.5). The TZ is not a fixed point and its position varies with age. The cervical pre-neoplastic lesion originates from the TZ and therefore this area is the target of the cervical screening.5
Figure 1.5: Squamocolumnar junction: This is the meeting point between squamous epithelium and mucin secreting columnar cells (Hematoxylin and eosin stain)
Ovaries
These two oval structures are 3–5 cm diameter each and are situated lateral to fallopian tubes and uterus. The ovary secretes female sex hormones estrogen and progesterone. The other important function of the ovary is production of mature ovum. Each ovary contains overall 400000 ova. The ovum comes out from the ovary at the time of ovulation and passes to the fallopian tube. If fertilization does not occur then this ovum is expelled from the uterus. The fertilized ovum is embedded in the endometrium.
Fallopian Tubes
Fallopian tubes are 9–11 cm long tubular structure that extend from the cornual ends of uterus laterally up to each ovary. Both the tubes directly open to the peritoneal cavity to receive the ovum from ovary.
CYTOLOGY
The normal cellular components of cervical smear are:
- Squamous cells
- Columnar cells
- Metaplastic squamous cell
- Endometrial cells
- Inflammatory cells
- Others
These cellular constituents are important to identify for the proper interpretation of the cervical smear.6
Squamous Cells
Superficial Squamous Cells
The superficial squamous cells are the predominant constituents of the cervical smear. These cells are scrapped from the outer most layer of the cervical squamous epithelium. These cells are large and polygonal in shape with abundant orangeophilic to green cytoplasm (Figure 1.6). The diameter of the cell is 30–45 micron and nucleus is about 5–7 micron. The cytoplasm of the cells often shows kerato-hyaline granules which are small dark brown in color in Papanicolaou's (PAP) stain (Figure 1.7). At times the air may be trapped in between the smear and cover slip and brownish granules like artifact may be created. This cell is labelled as “corn flakes” cell (Figure 1.8).
The characteristics of superficial cells are:
- Large polygonal cells
- Abundant thin pink to green cytoplasm
- Cytoplasmic orangeophilia is often seen which indicates intracellular keratin.
- Centrally placed small pyknotic nuclei.
Intermediate Cells
These cells are almost of same size and shape as that of superficial cells. They contain abundant greenish cytoplasm. The basic difference of superficial cells and intermediate cells lies in the nucleus. The nuclei are relatively large and vesicular in the intermediate cells compared to pyknotic nuclei in the superficial cells (Figure 1.6).
The characteristic features of intermediate cells include:
- Large polyhedral cells
- Abundant pale green cytoplasm
- Centrally placed vesicular nucleus.
Note: Intermediate cells with folded margins look like boat shaped. These cells are labelled as navicular cells. Navicular cells are seen in pregnant patients due to the progesterone effect on cervical epithelium.
Parabasal and Basal Cells
The parabasal and basal cells are smaller in size (Figure 1.9). The diameter of parabasal cell is 10–25 micron and nucleus is 6–8 micron. The nuclei of the cells are relatively large with homogenous bland chromatin. The number of parabasal cell increases as age increases.
The cytological features of parabasal cells are:
- Small round to oval cells
- Dense cytoplasm
Note: Basal and parabasal cells are frequently seen in atrophic smear of post-menopausal patient. The parabasal cells may also be increased in inflammatory smear. The cells may be mistaken as dysplastic cells as they have high nucleo-cytoplasmic (N/C) ratio. However, the nuclear chromatin is homogenous in the parabasal cells.
Changes of Squamous Epithelium
The following changes may occur in the squamous epithelium:
Hyperkeratosis
The term hyperkeratosis is characterized by the presence of thick keratin layer over the squamous lining of the cervix. In cervical cytology smear hyperkeratosis is represented by single or clusters of anucleated squamous cells (Figure 1.10). The nuclei of the cells may be only seen as clear central zone.1 Hyperkeratosis is a non-specific finding and may be seen in inflammation, post cryotherapy or uterine prolapse. However, hyperkeratosis may be an associated finding in Human Papilloma virus (HPV) infection.2
Parakeratosis
The term parakeratosis means the presence of nucleated squamous cells in keratin layer of the hyperkeratosis. On cytology smear the parakeratotic cell is characterized by superficial squamous cells with small, pyknotic or hyperchromatic nuclei2,3 (Figure 1.11). The presence of parakeratosis is also a non-specific finding. Probably the female with the presence of parakeratosis in the cervical smear has higher chance of HPV infection.
Pseudoparakeratosis
The term “pseudoparakeratosis” indicates the glandular cells with deep orangeophilic cytoplasm simulating parakeratotic cells. These type of cells are seen in patients who receive oral contraceptive. The pseudoparakeratotic cells may also be seen as degenerated parabasal cells in atrophic background.9
Figure 1.11: Parakeratotic cells: The cells with orangeophilic cytoplasm and dense pyknotic nuclei (see arrow)
Dysplastic cells
Discussed later.
Endocervical Cells
The endocervical cells are round to columnar in shape (Figure 1.12, 1.13). The cells may be present in small clusters with honey comb appearance (Figure 1.14) or they may be discrete or in small strips. The cells contain moderate amount of cytoplasm which may be vacuolated. The diameter of endocervical cell is about 18–20 micron and nucleus is 8–10 micron. The nuclei of the cell are round with finely granular chromatin having occasional tiny nucleoli. Occasionally the endocervical cells may be multinucleated.10
Figure 1.13: Endocervical cells: Higher magnification showing columnar cells with basally placed nuclei
The characteristic features of endocervical cells are:
- Small round to columnar cell
- Vacuolated cytoplasm and basally placed nuclei
- Round nuclei with small nucleoli and fine granular chromatin
- Honeycomb like arrangement is often seen
- Picket fence appearance in side view
Note: The relative frequency of endocervical cells are more in liquid-based cytology (LBC) due to use of the brush that samples cells from the endocervical canal. Endocervical cells with tuft of cilia from terminal plate are seen in tubal metaplasia (Figures 1.15 and 1.16). The cells should not be mistaken as dysplastic cells.
Metaplastic Squamous Cells
The endocervical cells may undergo squamous metaplasia. This is the commonest change in the endocervical epithelium. The immature parabasal cells are the main constituents of the metaplastic cells. The cells are geometrical shaped with projected cytoplasmic margin and are arranged in interlocking manner that gives a mosaic like pattern (Figures 1.17 and 1.18). The squamous metaplastic cells are relatively small with scanty cytoplasm. The nuclei are enlarged with slightly irregular contour and mildly hyperchromatic.
The cytological features of squamous metaplastic cells include:
- Relatively small with scanty dense cytoplasm resembling parabasal cells
- Cells have projected margin that resembles spider-leg
- Round nuclei with fine chromatin and small nucleoli
- Mosaic like pattern in clusters
Figure 1.17: Metaplastic endocervical cells: Cells with polyhedral appearance, small in size with cytoplasmic projections (see arrow)
Note: Metaplastic squamous cells should not be mistaken as dysplastic cells. The following features are helpful in distinguishing metaplastic cells from the dysplastic cells:
- Relatively small nucleus
- Regular nuclear margin
- Fine chromatin
- Low N/C ratio.
Whereas, the cells of SIL have higher N/C ratio, irregular nuclear margin and hyperchromatic nuclei.
Endometrial Cells
The endometrial cells can be normally found in women with reproductive age period. The presence of endometrial cells in post-menopausal patient is abnormal. The endometrial cells are usually present in small tight cohesive clusters (Figures 1.19 and 1.20). The cells show scanty cytoplasm and round dark nuclei. Nuclei may compress each other causing moulding. Single cell death or apoptotic cells may be seen in cluster of endometrial cells.
The salient cytological features of endometrial cells include:
- The cells are usually arranged in small tight spherical clusters
- Small round cells with scanty cytoplasm
- Round dark nuclei
- Nuclear molding
- No nucleoli.
Note: Benign endometrial cells may be seen in endometrial polyp, endometritis, endometriosis and abortion. It may also be present during menstrual period. The endometrial cells may be mistaken as: cells of high grade squamous intraepithelial lesion (HSIL), poorly differentiated squamous cell carcinoma and well differentiated adenocarcinoma.
Figure 1.19: Endometrial cells: Tight cohesive cells with scanty cytoplasm and round nuclei with condensed chromatin (see arrow)
Inflammatory Cells
Polymorphonuclear leukocytes and lymphocytes are frequently seen in cervical smear. Mere presence of inflammatory cells does not indicate inflammation.
Histiocytes
These cells are also normally found in the cervical smear. The cells are:
- Large cells
- Moderate cytoplasm
- Central kidney shaped nuclei
Note: These cells may be seen in various conditions such as pregnancy, foreign body reactions, and endometrial cancer.
Others
Spermatozoa
The cells can be mistaken as Candidal spores. Long tail of spermatozoa are easily identified in the smear (Figure 1.21).
Lactobacilli
Lactobacilli are gram positive rod shaped bacilli and are normally present in cervical smear (Figure 1.22).
Ova of Parasites
Rarely ovum of parasites are contaminated in cervical smears.
Giant Cells
The multinucleated giant cells are not uncommon in cervical smear (Figures 1.23 and 1.24). They are mostly nonspecific in nature. The differential diagnosis of multinucleated giant cells in the cervical smear include:
- Viral infection,
- Tuberculosis
- Syncytio and cytotrophoblasts
- Radiation exposure
- Squamous cell carcinoma and
- Choriocarcinoma.
CELLS OF ADJACENT ORGAN
Urothelial Cells
- Columnar cells with round regular nuclei
- Difficult to identify.
Colonic Cells
- Aggregates of columnar cells in rows.
Acellular Material
- Contaminants (Figure 1.25): Dust of surgical gloves and lubricant jellies etc.
Essential Information Needed for Interpretation of Cervical Cytology
Some information is mandatory before the interpretation of any cervical cytology smear, such as (Box 1.1):
- Patient's age: Mentioning the age of the patient in the clinical request form is mandatory. The diagnostic interpretation may vary according to age such as presence of endometrial cells above 45 years of age should be considered as abnormal.
- Date of last menstruation and the status of menopause: Menstrual history particularly the date of last menstruation may help to interpret the endometrial cell clusters around the menstrual period.17Similarly menopause may affect the pattern of cervical smear. Parabasal and basal cells are usually more in post-menopausal smear and may show reactive nuclear enlargement that can be mistaken as malignancy.
- Intrauterine contraceptive device (IUCD): The history of IUCD may help to recognize the “bubble gum” cells and IUD cells. These cells can be mistaken as the cells of adenocarcinoma.
- Hormone status: The history of hormonal replacement is essential to recognize the changes due to hormone.
- Previous history of squamous intraepithelial lesion (SIL) or histopathology: The previous history of SIL helps to search and recognize the dysplastic cells. It also helps to take decision to have further colposcopic or histopathological examination. Previous history of histopathology or cone biopsy is also necessary. The large number of ciliated endocervical cells are often seen after sampling from a cone biopsy.
- History of radiation or chemotherapy: Radiation or chemotherapy may cause cytomegaly having nuclear enlargement. It is easier to interpret such cells with proper history.
How to Recognize a Dysplastic Cell
In every smear a thorough search is needed to identify the dysplastic cells. The area of LBC smear is small and it is much easier to find out the abnormal cells. Moreover, relatively clean background free of mucus, necrotic tissue and 18inflammatory cells make the search process easier. One should always screen the smear thoroughly either horizontally or vertically in a zig-zag way. The essential characteristics of dysplastic cells are (Box 1.2):
Arrangement
The dysplastic cells are predominantly arranged singly (Figure 1.26). Occasionally the dysplastic cells may be arranged as tight cohesive cluster known as hyperchromatic crowded group of cells. The hyperchromatic crowded group (HCG) may be noted in both benign and dysplastic cells (Figure 1.27). Irregular margin of the cluster and the loss of polarity of the peripheral cells are the features of HCG produced by dysplastic cells.
Chromatin Pattern Alteration
Alteration of the chromatin pattern of the cell is the most important characteristic to recognize a dysplastic cell. Normal cells show homogenous bland chromatin. The dysplastic cells show: speckled chromatin, fine granular, even or irregular coarsely granular chromatin (Figures 1.28, 1.29 and 1.30). The type of change of chromatin pattern has little effect on grade of dysplasia.
Hyperchromasia
The majority of the dysplastic cells show hyperchromatic nuclei. The hyperchromatic nuclei are dark colored and should always be differentiated from pyknotic nuclei or nuclei with smudged chromatin.
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Rarely dysplastic cells are hypochromatic and pale in color (Figure 1.31). The pale dyskaryotic cells show abnormal chromatin pattern.
Nuclear Enlargement and N/C Ratio
Simple enlargement of nucleus along with excess cytoplasmic area may not be significant. Nuclei are relatively large in parabasal and endocervical cells. However, increased nucleo-cytoplasmic (N/C) ratio is pathognomonic of dysplastic cells (Figure 1.32).
Nuclear Contour
Nuclear margin irregularity is also one of the characteristic features of dysplasia. There may be minor irregularity of the nuclear margin in the cells of low grade squamous intraepithelial lesion (LSIL) and grossly irregular nuclear contour in HSIL (Figure 1.32).21
Figure 1.32: High nucleocytoplasmic ratio and irregular nuclear membrane in dysplastic cells (see arrow)
The alteration of nuclear margin is usually more evident in LBC preparation as the cells are better fixed in liquid-based cytology and the air drying is completely avoided. However, the nuclear margin may be completely regular in dysplastic cells.
Thickened Nuclear Membrane
Nuclear membrane is often thickened in dysplastic cells (Figure 1.33). This is also an important feature to recognize dysplastic cells. Thickened nuclear membrane is more frequent in HSIL and is more evident in LBC.22
REFERENCES
- Izadi-Mood N, Sarmadi S, Alijani S, Sanii S. The significance of hyperkeratosis in pap smears with squamous intraepithelial lesion. Acta Cytol. 2012;56(4):379–82.
- Zahn CM, Askew AW, Hall KL, Barth WH Jr: The significance of hyperkeratosis/parakeratosis on otherwise normal Papanicolaou smears. Am J Obstet Gynecol 2002;187:997–1001.
- Kern SB. Significance of anucleated squames in Papanicolaoustained cervicovaginal smears. Acta Cytol. 1991;35:89–93.