Vaginal microflora analysis

General clinical examination of vaginal material

The vaginal discharge is examined to assess the nature of the microflora and identify the inflammatory process, as well as to identify atypical cells and assess the production of sex hormones ("hormonal mirror"). Material for cytological diagnostics is obtained in various ways: by aspiration and scraping of the contents of the posterior vaginal fornix, cervical canal, or by obtaining smears of imprints.

Vaginal microflora

In diagnostics of inflammatory processes of the female genital tract, the study of the microflora of the discharge plays a crucial role. From a modern standpoint, the normal microflora of the genital tract is considered as a set of microbiocenoses occupying numerous ecological niches on the skin and mucous membranes. Microorganisms that make up the normal microflora of the vagina are in various relationships with each other (neutralism, competition, commensalism, synergism, parasitism, etc.). A change in the number of a particular type of microorganism in the corresponding biotope or the appearance of bacteria that are not typical for this habitat serves as a signal for reversible or irreversible changes in the corresponding link of the microecological system. A feature of the normal microflora of the genital tract in women is its diversity.

Facultative lactobacilli predominate in the vaginal contents of women with a regular menstrual cycle and pregnant women, but are virtually absent in prepubertal girls and postmenopausal women. The number of lactobacilli in the vagina of healthy women is 10 ⁵ -10 ⁷ CFU/ml. Estrogen production in women of reproductive age increases the glycogen content in the vaginal epithelium. Glycogen is metabolized into glucose and then, with the help of lactobacilli, into lactic acid. It provides a low pH level (less than 4.5), promotes the growth of acidophilic microorganisms, in particular lactobacilli. In addition to lactobacilli, the vaginal biocenosis includes more than 40 species of other bacteria, but their share does not exceed 5% of the total number of microorganisms. In healthy non-pregnant women, the rank sequence of bacterial species is as follows: lactobacilli, bifidobacteria, peptococci, bacteroides, epidermal staphylococci, corynebacteria, gardnerella, mobilungus, mycoplasma. The ratio of anaerobic to aerobic flora is 10:1.

Species composition of normal vaginal microflora

Microorganisms	Content, frequency of detection
Total number of microorganisms	10 5 -10 7 /ml
Facultative lactobacilli	More than 90%
Other microorganisms:	10%
Staphylococcus epidermidis	36.6%
Bifidobacteria	50%
Candida albicans	25% (in pregnant women up to 40%)
Gardnerella vaginalis	40-50%
Ureaplasma hominis	70%
E. coli	In small quantities
Staphylococci and streptococci	In small quantities
Anaerobic microflora (bacteroids, peptostreptococci, clostridia)	In small quantities

Normal bacterial flora plays an antagonistic role, preventing the invasion of pathogenic microorganisms, and any invasion into healthy epithelium is almost always accompanied by changes in the vaginal microflora.

To assess the state of the vaginal microflora in clinical practice, a bacteriological classification of 4 degrees of purity has been used for a long time, taking into account the number of lactobacilli, the presence of pathogenic bacteria, leukocytes, and epithelial cells.

• I degree. Smears contain epithelial cells and a pure culture of facultative lactobacilli. The reaction of the vaginal contents is acidic (pH 4-4.5).
• II degree. A small number of leukocytes, fewer facultative lactobacilli, other saprophytes are present, mainly gram-positive diplococci, the reaction of the contents remains acidic (pH 5-5.5).
• III degree. Large number of epithelial cells, leukocytes. Facultative lactobacilli in small quantities, diverse coccal flora; the reaction of the contents is slightly acidic or basic (pH 6-7.2).
• IV degree. Epithelial cells, many leukocytes, diverse pyogenic flora with a complete absence of vaginal bacillus, basic reaction (pH above 7.2).

At present, the conventionality of this classification and its insufficient informativeness are obvious. It does not take into account the diversity of types of normal microflora, their relationships, as well as the possible presence of pathogenic agents such as gonococci, trichomonads, fungi, chlamydia, etc.

Violation of the ratio of the content of different types of microorganisms or the species composition of their associations leads to the occurrence of inflammatory processes in the vagina. The mechanisms that change the normal ecosystem of the vagina include: hormonal factors that determine the content of glycogen in epithelial cells; microbial antagonism; immune system disorder; sexual behavior.

For the correct interpretation of pathological changes in inflammatory processes in the female genital tract, knowledge of the cytomorphological features of the normal vaginal mucosa is important.

The vaginal epithelium (stratified squamous) undergoes cyclic changes during the menstrual cycle under the influence of sex hormones. The following layers can be distinguished in the stratified squamous epithelium of the vagina: superficial, intermediate, external basal and internal basal. In the first days after menstruation, approximately one third of the vaginal epithelium remains, then during the menstrual cycle it is restored again.

Four types of epithelial cells are distinguished in vaginal smears.

• The cells of the superficial layer are large (35-30 µm) polygonal in shape, the nucleus is small (6 µm), pyknotic. The cells are often located separately. These cells are present in large quantities from the 9th to the 14th day of the menstrual cycle.
• The cells of the intermediate layer are smaller in size (25-30 µm), irregular in shape, the nucleus is larger, round or oval. The cells are often arranged in layers. They are present in all phases of the menstrual cycle.
• Parabasal layer cells are small in size, round in shape, with a large round central nucleus. They are present in small numbers only during menstruation and appear in smears during menopause or amenorrhea.
• Basal (or atrophic) cells are smaller than parabasal cells, round in shape, with a large nucleus and a nucleus-to-cytoplasm ratio of 1:3. They appear during menopause and postpartum amenorrhea.

In addition to epithelial cells, vaginal smears may contain erythrocytes (involved with minor tissue damage), leukocytes in the amount of 6-8, and after ovulation up to 15 in the field of view, they enter the discharge either by migration through the vaginal wall, or as a component of the inflammatory exudate.

The mucous membrane of the cervical canal is covered with high prismatic epithelium with basal arrangement of nuclei, the cytoplasm of the cells contains mucus. Reserve (combination) cellular elements are often found under the prismatic epithelium. Two types of epithelium - multilayer flat and prismatic - contact in the area of the external cervical os. In smears, prismatic epithelial cells, single metaplastic cells, and mucus are normally found (there may be a lot of leukocytes in the mucous plug - up to 60-70 in the field of vision).

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