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Microscopic examination of exfoliated cells from the vagina of the intact bitch is used to assist in determining the stage of the heat cycle. Staging of the heat cycle assists in determining the optimal time for breeding and to screen for ovarian remnant in spayed females exhibiting signs of estrus. This article will review indications, limitations, sample collection, and evaluation of vaginal cytology smears for estrus cycle staging.
Vaginal cytology is an imprecise predictor of ovulation because peak cornification can occur any time from six days before to three days after the LH surge. Many reproductive specialists use vaginal cytology primarily to determine when to start submitting serum for progesterone levels and to determine the first day of diestrus. Progesterone and LH concentrations more precisely predict ovulation for timing of insemination, as described above.
Theriogenologist Dr. Tessa Fiamengo demonstrates proper vaginal cytology sample collection technique in this video.
The cells are then transferred to a glass microscope slide by gently rolling the swab with minimal pressure to decrease cell rupturing. Slides are air dried and stained with a Romanowsky-type stain such as Diff-Quik®.
Stages of the estrous cycle: Stages of the canine estrous cycle include proestrus (mean duration of nine days; range of two to 17 days), estrus (mean duration of nine days; range of three to 21 days), and diestrus (typical duration of 57 days in the pregnant bitch and a range of 57–90 days in the non-pregnant bitch). Anestrus is the “rest” period between heat cycles and has a wide range from 2–10 months (mean duration of six months). Vaginal cytologic findings in each of the stages and anestrus will be reviewed.
Vaginal Cytology Cell Types: Squamous epithelial types from least mature to most mature including parabasal cells, intermediate cells and superficial cells (aka, cornified cells) are shown in Figure 1. Other cell types commonly found in vaginal cytology smears include erythrocytes or red blood cells (RBCs) (Figures 3, 6, 7, 9, 10) and neutrophils (Figures 2, 5, 10). Bacteria may be found in any stage and some are noted to be phagocytized by neutrophils (Figures 2–7 and 9–10). The finding of phagocytized bacteria must be differentiated from bacterial vaginitis. Metestrum cells are vaginal epithelial cells with neutrophils in their cytoplasm (Figure 1F). They are not specific for any stage of the cycle and can be observed whenever neutrophils are present (most commonly in proestrus and diestrus). Sperm may be observed in a bitch that has been bred or inseminated recently (Figure 8).
Anestrus: Parabasal and intermediate cells are the predominant epithelial cell types present in the inter-estrus period (Figure 2). RBCs are typically absent. Neutrophils and bacteria may be present in low numbers. Progesterone concentration in anestrus is typically less than 1.0 ng/mL and often less than 0.5 ng/mL.
Proestrus: The rise in estrogen as follicles mature, which starts in proestrus, causes the vaginal epithelium to proliferate and RBCs to pass through uterine capillaries. In early to mid-proestrus, cytology findings include a mix of epithelial cell types (parabasal, small and large intermediate cells) along with variable numbers of erythrocytes and neutrophils (Figures 3, 4). The progesterone concentration usually remains below 1.0 ng/mL. In late proestrus when estrogen is at or near peak levels, large intermediate and superficial cells predominate (Figure 5) and neutrophils may be absent. Bacteria are often present in variable to large numbers throughout proestrus. As estrogen is decreasing towards the end of proestrus, the LH surge occurs in late proestrus or early estrus. The progesterone concentration typically increases to 2 ng/mL near the time of the LH surge.
Estrus: Superficial cells reach >80% of the epithelial cell population in estrus (Figures 6–8). RBCs often decrease and neutrophils are typically absent or present in very low numbers. High numbers of bacteria are generally present in the background and on the surface of the superficial cells. Ovulation occurs approximately 48 hours after the LH surge. Progesterone concentration near the time of ovulation averages 5.0–6.0 ng/mL (range: 4.0–10.0 ng/mL) with a rapid rise to 15–40 ng/mL by the end of estrus. Estrogen concentration continues to decrease during estrus. Because maximal cornification (near 100% superficial cells) can occur anywhere from six days before to three days after LH peak, vaginal cytology is not an accurate predictor of ovulation timing. Progesterone testing should be initiated by 5–7 days after the start of vaginal discharge or no later than the beginning of cytologic estrus, if timing ovulation.
Diestrus: Diestrus occurs approximately eight days (range of 6–10 days) after the LH peak. This stage features elevated progesterone concentration, regardless of whether the bitch is pregnant or not, and much lower estrogen levels following the receptive estrus stage. Progesterone concentration peaks in mid-diestrus in the 15–90 ng/mL range and gradually decreases after that to <1.0 ng/mL if the bitch is not pregnant. In pregnant bitches, the progesterone concentration remains above 2.5 ng/mL until dropping abruptly just before whelping. With the decreasing estrogen level, vaginal epithelium decreases in thickness resulting in an abrupt change in cytologic appearance on the first day of diestrus. Superficial cells decrease to 20% or less and parabasal and intermediate cells increase (Figures 9, 10). Neutrophils return in low to high numbers and both extracellular and phagocytized bacteria are often present in normal cycling bitches. RBCs may or may not be present. There is overlap in the appearance of proestrus and diestrus, thus without evaluation of sequential vaginal smears, it may be challenging to accurately determine the stage.
Vaginal cytology is useful for monitoring progression from proestrus to estrus, but is an imprecise predictor of ovulation because peak cornification can occur any time from six days before to three days after the LH surge. When superficial epithelial cells represent approximately >80% of the vaginal epithelial cells (cytologic estrus) or when the bitch has reached day 5–7 of vaginal discharge, monitoring of serum progesterone levels is initiated to determine the optimal time for breeding. Additional information can be obtained by determining the LH surge in serum samples, which more precisely predicts the ovulation date than progesterone levels alone. Vaginal cytology may also be used to determine the first day of diestrus along with ovulation timing using LH levels and/or progesterone for predicting the whelping window. Vaginal cytology may be a useful screen for ovarian remnant syndrome in some bitches. Cytologic evidence of vaginal cornification suggests estrogen influence, which prompts additional diagnostic tests for ovarian remnant, such as reproductive ultrasound examination, MRI and/or hormone tests (progesterone and anti-Mullerian hormone test).
