Vaginal cytology is an easy and cheap method with great value in small animal reproduction. As a general introduction in function of cellular types that we can see, we can determine the phase of the cycle in the bitch or queen. First we will describe the types of cells that we have and then the correlation with the ovarian cycle. The alterations in % and types of cells are directly related to serum estrogen concentration. Estrogens are the hormones secreted by a maturing follicles and cause a thickening of vaginal linin, moving the cells from her blood supply, with the subsequent degeneration and atrophy. Thus, vaginal cytology is always a reflex of estrogen activity on the female.
Techniques in vaginal smears
The preferred method to obtain a good vaginal cytology, is the application of a cotton swab in the cranial vagina of a bitch . The lip of the vulva are gently separated with the fingers and a cotton swab is passed into the dorsal aspect of the vulva. We must avoid clitoris fossa and advance in a craniodorsal direction, toward vertebral column. At this point we rotate the cotton swab and withdraw it. We must observe the color, and type of secretions and rotate gently the cotton tip in a slide. Usually three or four slides are fixed every time, with the name and date. A lot of stains has been described, but usually Diff-Quick, new methylene blue and Schor's stain modification (with criteria for keratinization) are used.
In the queen the cotton swab must be of adequate size (urethral human culturette tube) and introduced only 1 to 1,5 cm in the vagina. The main differences between queen and bitch is the relatively straight horizontal anatomy of the vestibule of the vagina, the vagina, and the cervix. Usually when we introduce the cotton swab we are inducing the ovulation of queen (cervico-hypothalamus reflex) with the subsequent external behaviour aspect (vocalization and others).
Type of cells that we can see in a vaginal cytology
We can classify the cells in keratinized versus nonkeratinized or cornified versus noncornified. The cornification or keratinization is a reflex of circulating estrogens. The basal cells (near blood supply) are small, round and with a clear nucleus, but in the cornified cells (with no blood supply) the nucleus become smaller, pyknotic and finally is disintegrated leaving an anuclear cell.
We will describe the principal characteristics of different types of cells:
Parabasal cells: small, round, slightly oval, large vesiculated nucleus and small cytoplasm. Good staining.
Intermediate cells: slightly larger than parabasal cells to twice that size. Smooth, oval to rounded irregular borders, nucleus smaller than in parabasal cells. More cytoplasm than parabasal cells.
Superficial cells: Are dead cells, are the largest in vaginal cytology with a sharp, flat, angular cytoplasmic borders and a small pyknotic, fading nuclei or without nuclei.
Anuclear squames: irregular vaginal cells, no nucleus, smaller than superficial. These are the cells that have also been called "fully cornified."
Metestrus cells: Large intermediate vaginal cells that appear to have one or more neutrophils in their cytoplasm. Are typical of bitch diestrus.
Foam cells: parabasal and intermediate cells with obvious cytoplasmic vacuoles. May be associated with diestrus and anestrus.
Neutrophils: Inflammatory cells that can be normal or abnormal in vaginal cytology in function of the estrus period
Red blood cells: Blood can be normal in the bitch but always is abnormal in the queen.
Correlation between ovarian cycle and vaginal cytology
In the bitch
Dogs has two ovarian cycles twice yearly, in spring and again in autumn, however some bitches experience more or less than two cycles a year. These are in function of breed, climate, and others.
The bitch ovarian cycle is divided in four phases: proestrus, estrus, diestrus and anestrus. We can correlate this phases with vaginal cytology.
Proestrus: Period of high follicular activity. The female attracts the males but is not receptive to the copula, the vagina has a bloody discharge and is edematous. The duration of this phase is usually from 6 to 11 days, but the average can be from 2-3 days to 25 days. The vaginal cytology is typical of estrogens secreted by developing ovarian follicles with RBC, numerous parabasal, small and large intermediate vaginal cells, neutrophils are common, bacteria, and a dirty background in the early proestrus and with no neutrophils, less blood cells, more than 80% of superficial cells and a clear background in the late proestrus. This late proestrus is difficult to distinguish from early estrus.
Estrus: In this phase the bitch allows the mating. Estrus starts with this allowing and ends when she no longer accepts the male. Another criterion to define the finish of estrus is the vaginal cytology start of diestrus. Estrogen concentrations reach a peak 1 or 2 days prior to the onset of estrus, and during estrus starts the decline in circulating estrogens and the start in progesterone secretion by luteinized follicular cells first and corpus luteum after. The vaginal cytology is relatively constant with superficial cells and anuclear squames accounting more than 80% of the total vaginal cells and often reaching 100%. No neutrophils must be present and red blood cells are not usually presents.
Diestrus: Is the phase with predominance of progesterone secretion with or without pregnancy. Vaginal cytology is characteristic in this phase with a dramatic "shift" form a estrus with a 80-100% superficial cells to one of 80-100% of parabasal and intermediate cells plus neutrophils and metestrus cells. This change is produced in about 24-48 hours.
Anestrus: Is defined as the period of sexual arrest. The uterus involution is produced and start with whelping and ends with a new proestrus. In non pregnant bitches the beginning of anestrus is not readily easy to distinguish clinically with no obvious demarcation between diestrus and anestrus.
In the queen
Queen reach the sexual maturity after she attains at least 80% of adult body weight and if the photoperiod is appropriate (usually between 6 and 9 months of age). By definition the cat is seasonally polyestrous. She cycles repeatedly throughout a breeding season unless the cycle is interrupted by pregnancy, ovulation, illness or pseudogestation. This is a important difference with the bitch, the influence of photoperiod and hours of daylight.
The phases of the queen sexual cycle are the same that in the bitch, but with some differences. Usually we aggregate proestrus and estrus, because they are difficulty distinguished. Another characteristic of the queen is that she has a interestrual period, between estrus, in the adequate photoperiod season with a typical changes in the vaginal cytology. A typical cycle of a queen is: anestrus-follicular phase (proestrus + estrus)-interestrus-follicular phase ....... - anestrus
Another important characteristic in the physiology of the queen, is that is a ovulation induced female. When the tom introduce the penis in the queen vagina, he induce the ovulation reflex. In some occasions a female can ovulate without copula.
Proestrus: Is the period of follicular function after anestrus or interestrus period, estrogen synthesis, secretion and changes in behaviour and vaginal cytology. The behavioral changes are rubbing, vocalizing, lordosis, and rolling.
Estrus: Is associated with follicular estrogen synthesis and secretion. Usually is associated with proestrus as a "follicular phase."
Vaginal cytology in "follicular phase": clear background, without cellular debris and easy visualization of vaginal cells. The squames increase to 10% of the total on the firsts days reaching 40% at seven days, and then slowing again to 10%. In this phase the intermediate cells decrease from 40-50% in the interestrus period to 10% during follicular phase. No parabasal cells, no RBC, and no PMNN are typical of this phase.
Interestrus: follows cessation of follicular activity and the suppression of estrogens secretion. In this phase the behaviour is normal for the queen without rubbing or vocalization. The vaginal cytology is really curious and has a lot of typical characteristics: we can observe all types of vaginal cytology cells, intermediate, squames, parabasal and superficial and again we can see debris in the background.
Diestrus: As in the bitch is the phase with progesterone dominance. The corpus luteum produce progesterone after ovulation (induced ovulation in the queen).
Anestrus: Again, anestrus is defined as a period of clinical reproductive quiescence. The female do not attract males and not display sexual behaviour. In the vaginal cytology we have low cellularity with all possible types of cells and a dirty background with debris.
Clinical usefulness of vaginal cytology
We will see with practical cases the normal and possible applications of vaginal cytology as:
Management of normal breeding
Shipping or receiving a bitch
Study of unusual cycles
Predicting whelping dates
Diagnosis of vaginal problems, follicular problems and uterine diseases
Application of reproductive drugs: abortifacients
Limitations of vaginal cytology
Vaginal cytology doesn't permit gestation diagnosis, doesn't permit identify ovulation day, and is just a complement of a good clinical examination, anamnesis and hormone assays.