The Canine Estrous Cycle: Staging Using Vaginal Cytological Examination

Valerie R. Beimborn, DVM; Heather L. Tarpley, DVM; Perry J. Bain, DVM, PhD; and Kenneth S. Latimer, DVM, PhD

Class of 2003, Ross University, School of Veterinary Medicine, St. Kitts, West Indies (Beimborn) and Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388 (Tarpley, Bain, Latimer)

Introduction

Vaginal cytology is a simple technique that can be used by practitioners to help characterize stages of the reproductive cycle of the bitch or to evaluate certain diseases of the genital tract. Vaginal cytology usually is used in conjunction with the physical examination, clinical history, vaginoscopy, and hormonal assays to determine the stage of the reproductive cycle. This is especially important if artificial insemination is to be performed. Other uses of vaginal cytology and ancillary testing include determination of the whelping date, diagnosis of inflammation of the vagina, and identification of some types of neoplasia involving the vaginal vault and urethral orifice.

Estrous Cycle of the Bitch

The estrous cycle of the bitch contains four distinct stages that can be followed cytologically (Table 1). These stages include proestrus, estrus, diestrus, and anestrus. For some simplification and indication of which structures predominate throughout the cycle, these four stages may be grouped into the follicular phase (proestrus and estrus) or the luteal phase (diestrus and anestrus). The bitch frequently has been classified as monocyclic or having a reproductive cycle that lacks frequent, recurring periods of heat and has a long period of anestrus regardless of pregnancy status.

Table 1. Characteristics of the Canine Estrous Cycle

Cycle Stage	Length	Hormonal changes	Predominant Cell Types	Erythrocytes	Neutrophils	Behavior of the Bitch	Clinical Signs
Proestrus	~9 days	Estrogen	Early: mixed population of cells Late: large intermediate and superficial cells	Early: present   Late: ±	Early: present   Late: decreased	Attractive to the male, but won’t stand for mating	Vulvar edema and swelling, bloody discharge
Estrus	~9 days	LH (surge) Estrogen Progesterone	90% superficial cells Bacteria usually present	±	Absent	Accepts male and will stand	Less edema, discharge becomes clearer
Diestrus	~60 days	Progesterone	Abrupt change in relative # of epithelial cells. Superficial cells decrease by 20%	±	Few to none	Ceases to accept male	Little discharge, edema decreased
Anestrus	Depends on whether pregnant or not. 70-80 days for CL to regress and 130 days to repair endometrium.	Progesterone	Parabasal and Intermediate cells	Few if any	Few if present	No outward signs	Scanty, tenacious secretions

Proestrus: Proestrus typically lasts about 9 days. This is the reproductive phase that immediately precedes estrus. Estradiol concentration increases as ovarian follicules mature and the uterus enlarges. The vaginal epithelium proliferates accompanied by diapedesis of erythrocytes from uterine capillaries. Thus, erythrocytes account for most of the cells observed on vaginal smears at this reproductive stage. Grossly, vulvar edema, hyperemia, and a serosanguineous discharge occur.^7,8

Estrus: Typically, estrus lasts for about 9 days. Estrus is characterized by the period of acceptance of the male by the bitch. The female will seek out males and stand for them prior to copulation. This stage of the reproductive cycle is difficult to determine cytologically because distinguish features are only present microscopically at the beginning and the end of estrus. During estrus, glandular secretions increase, the vaginal epithelium becomes hyperemic, and ovulation occurs. This stage of the reproductive cycle is influenced mainly by estrogens. The interval between successive estrus cycles is typically seven months.

Diestrus: This is the period where corpora lutea (CL) are present. The CL are fully functional and secrete increased large quantities of progesterone. During this phase of the reproductive cycle, the bitch refuses to accept the sexual advances of the dog. Physiologically, the uterine glands undergo hypertrophy and hyperplasia. The cervix constricts and vaginal secretions become tenacious. The length of diestrus is variable; however, regression of CL requires approximately 70-80 days.

Anestrus: Anestrus is a prolonged period of sexual rest where the reproductive system is quiescent. Secretions are minimal and very tenacious or absent. The cervix is tightly constricted and the vaginal mucosa is pale.

Collection and Preparation of Cytological Specimens

Vaginal cytology specimens are easily and quickly collected and prepared. A saline moistened cotton swab is inserted through the vulvar lips into the vagina. The swab is angled craniodorsally to avoid the clitoral fossa. A clean otoscope may be used to guide the swab and to provide a light source for visual guidance. Once the swab is located cranial to the urethral orifice, the swab is rotated slightly to exfoliate and collect the cells.

After the swab is removed from the vagina, the cells are transferred to a clean glass slide by rolling the swab along the surface of the slide. Intact cells are obtained during transfer by rolling the swab. In contrast, back and forth smearing of the swab to transfer the cells will cause sever cellular distortion and rupture. Once the cells have been transferred, the slides are air dried, stained with Romanowsky or new methylene blue stains, and examined microscopically.

An alternative method to collect cytologic material from the vagina is to use a plastic medicine dropper and saline solution. The dropper is passed into the vagina and the bulb is squeezed several times. Exfoliated cells are collected by saline lavage. A drop of fluid is placed on a glass slide and a blood-type smear is made, air dried, and stained as above. The medicine dropper technique is more gentle but the saline saline will distort the morphology of the cells and dilute the cell count.

Various stains have been used for vaginal cytology including Romanowsky stains (Wright, Giemsa, Leishman, Diff-Quik®), new methylene blue, toluidine blue, trichome stains (Papanicolaou, Shorr, Sano) and hematoxylin and eosin (H&E). ¹Personal experience, preference, cost, and ease of use usually determine the choice of stain. In any event, the stain should be economical, easily stored, and produce consistent staining that is adequate for cytologic needs.⁶

Cytologic Features of Vaginal Epithelial Cells

The vaginal epithelium is influenced by hormonal changes during the estrous cycle, allowing cytologic monitoring of the various reproductive stages. The morphologic features of the epithelial cells are described below

Basal Cells: Basal cells are the youngest cells of the vaginal epithelium and serve as precursors of the other epithelial cell types. They are located along the basement membrane and rarely are seen in exfoliative cytology. These cells have a high nuclear to cytoplasmic (N/C) ratio, a round nucleus, and a small amount of basophilic cytoplasm.

Parabasal cells: These cells are the smallest cells vaginal epithelial cells seen in cytologic preparations. Parabasal cells also have a high N/C ratio, round nucleus, and basophilic cytoplasm. They are uniform in size and shape. Parabasal cells also may contain cytoplasmic vacuoles and are known as foam cells. The function of the vacuoles in these cells is unknown. Large numbers of foam cells may be found in prepubertal samples and should not be confused with neoplastic cells.

Intermediate cells: These cells vary in size but are usually two times the size of parabasal cells. Their N/C ratio is decreased and they have large amounts of blue-green, keratinized cytoplasm. Their borders are round to irregular and folded. Intermediate cells also may be subclassified as superficial intermediate or transitional intermediate cells according to their stage in the reproductive cycle.

Superficial cells: Superficial cells are the oldest vaginal epithelial cells. They either have small, round, pyknotic nuclei or lack a nucleus if they are cornified. Their cytoplasm is abundant, blue-green, and keratinized. Cell margins are angular with folded edges.

Cytologic Staging of the Canine Reproductive Cycle

Each stage of the canine reproductive cycle has a distinct, predominant cell population. that is easily identified cytologically.

Proestrus: Predominant cell populations in early to mid proestrus include nondegenerate neutrophils and a mixture of parabasal, intermediate, and superficial epithelial cells (Fig. 1). The background of the slide contains an abundance of mucus. Variable numbers of extracellular bacteria represent normal flora. In late proestrus, the number of neutrophils declines and superficial cells begin to predominate (Fig. 2).

Figure 1. Early proestrus with a mixture of nondegenerate neutrophils; parabasal, intermediate, and superficial epithelial cells, erythrocytes, and mucus (Wright-Leishman stain).	Figure 2. Late proestrus with erythrocytes, scattered superficial cells, and absence of neutrophils (Wright-Leishman stain).

Estrus: In estrus, the cell population consists of ~ 90% superficial cells and < 5% parabasal or intermediate cells (Fig. 3). Less mucus is present in the background of the preparation. Normal bacterial flora usually is present and organisms often are attached to the superficial cells.

Figure 3. Estrus characterized by superficial cells that are keratinized, largely anucleate, and have angular, folded cell margins (Wright-Leishman stain).

Diestrus: Cell populations change abruptly in diestrus (Fig. 4). Superficial cells decrease by 20% and smaller intermediate cells increase in number. Neutrophils often reappear and may contain phagocytosed erythrocytes and bacteria. Cytologically, this stage of the reproductive cycle can look very similar to early proestrus. Therefore, serial cytologic sampling is required to make the distinction.

Figure 4. Diestrus characterized by a decrease in superficial cells, increase in smaller intermediate cells, and presence of neutrophils. Cytologically, diestrus may appear similar to early proestrus (Wright-Leishman stain).

Anestrus: Parabasal and intermediate cells predominate during anestrus. Superficial cells are not present. Erythrocytes and neutrophils may be present in low numbers or are absent. ¹⁰

Vaginitis

Vaginitis (vaginal inflammation) may occur in bitches regardless of age, breed, or reproductive status. Clinically, the most common signs of inflammation are mucoid to purulent vulvar discharge, pollakiuria, and licking of the vulva.⁵ Typically, vaginitis is secondary to noninfectious causes such as vaginal anomalies (e.g,. strictures, persistent hymen, vaginal hypoplasia, double vagina), vaginal immaturity (i.e,. puppy vaginitis), chemical irritation from urine, or mechanical irritation (e.g, .foreign bodies, vaginal neoplasia).⁹ Primary bacterial infection of the vagina is less common. Occasionally, it can be difficult to distinguish pathogenic bacteria from normal vaginal flora. However, a heavy growth of a uniform bacterium is more suggestive of a pathogen.³ Infectious agents that may be observed infrequently include Brucella canis, Ureaplasma, Mycoplasma, and herpesvirus.

Cytologic changes in vaginitis include the presence of many degenerate or nondegenerate neutrophils (Fig. 5). Mucus, lymphocytes, and macrophages also may be present and may indicate chronicity. If vaginitis is due to an infectious agent, phagocytosed bacteria or intracnuclear inclusions also may be observed. An abundance of neutrophils may be present in diestrus. Therefore, this normal stage of reproduction must be distinguished from inflammation.²

Figure 5. Vaginitis demonstrating the presence of many nondegenerate to degenerate neutrophils and scattered epithelial cells (Wright-Leishman stain).

Vaginal Neoplasia

Vaginal neoplasia is the second most common form of reproductive neoplasia in the bitch, following mammary neoplasia.⁶ Vaginal neoplasia includes transitional cell carcinoma (TCC) of the urethra, transmissible venereal tumor (TVT), squamous cell carcinoma (SCC), leiomyoma, and fibropapilloma. Vaginal tumors are usually found in the older bitch except for TVT which is more common in young dogs. The mean age of occurrence of vaginal neoplasia in the bitch is ~10 years. ⁶

Bitches with vaginal neoplasia typically present because of a mass protruding from the vulva, vaginal discharge and dysuria, stranguria, tenesmus, urinary incontinence, or perineal swelling due to mechanical interference by a tumor of neighboring structures.^6,9 These tumors are diagnosed by a combination of physical examination, medical history, palpation (digital examination), vaginoscopy, vaginal cytology, and surgical biopsy. Vaginal cytology is most helpful in diagnosing TVT, TCC, and SCC.

Transmissible venereal tumor is transmitted sexually, by mucous membrane contact (i.e,. sniffing and licking), or via bite wounds and tends to be common in areas where dogs roam freely. These neoplasms are either benign or malignant in behavior (please see Clerkship paper Canine Round Cell Tumors for more information on this neoplasm). Metastasis is rare but may involve regional lymph nodes, skin, and subcutanteous tissues. TVT cells have a round nucleus, single nucleolus, and a thin rim of light blue, vacuolated cytoplasm (Fig. 6). TVTs may resolve spontaneously without treatment.⁶ Treatment with vincristine or irradiation is very effective. ¹¹

Figure 6. TVT cells with a round nucleus, single nucleolus, and a thin rim of light blue, vacuolated cytoplasm (Wright-Leishman stain).

Transitional cell carcinomas and SCC are common in the distal urethra of the bitch and may involve the vagina or vestibule or both. Digital examination, vaginoscopy, and surgical biopsy are best for a definitive diagnosis. Occasionally, fine-needle aspirates may provide a definitive diagnosis of TCC or SCC. Cells in TCC exhibit anisocytosis, anisokaryosis, and have an increased N/C ratio (Fig. 7; please see Clerkship paper Cytologic Diagnosis of Transitional Cell Carcinoma in Dogs and Cats for more information). Neoplastic squamous cells are characterized by anisocytosis, anisokaryosis, and waxy blue cytoplasm, occasionally with a perinuclear halo (Fig. 8) Cytologic evaluation of urine sediment is helpful but is only diagnostic in ~ 40% of cases. ⁶

Figure 7. Transitional cell carcinoma. The epithelial cells exhibit anisocytosis, anisokaryosis, nucleolar rings, basophilic cytoplasm, and have an increased N/C ratio (Wright-Leishman stain).	Figure 8. Squamous cell carcinoma. The neoplastic squamous cells exhibit anisocytosis, anisokaryosis, and waxy blue cytoplasm. Neutrophils and erythrocytes also are present (Wright-Leishman stain).

References

1. Baker R, Lumsden JH: Color Atlas of Cytology of the Dog and Cat. St. Louis, Mosby, Inc., 2000; p. 235-237.

2.Cowell RL, Tyler RD; Meinkoth, J.H.: Diagnostic Cytology and Hematology of the Dog and Cat. 2 ed. St. Louis, Mosby, Inc., 1999, Chapter 20. Veterinary Clinics of North America: Small Animal Practice, May 1991; 21(3): 523-531.

3. Freshman JL: Clinical approach to infertility in the cycling bitch. Vet Clin N Am: Small Anim Pract 21:427-435, 1991.

4. Goodman M: Ovulation timing: Concepts and controversies. Vet Clin N Am: Small Anim Pract 31:219-235, 2001.

5. Johnson CA: Diagnosis and treatment of chronic vaginitis in the bitch. Vet Clin N Am: Small Anim Pract 21:522-531, 1991.

6. Manothaiudom K, Johnston SD: Clinical approach to vaginal / vestibular masses in the bitch. Vet Clin N Am: Small Anim Pract 21:509-519, 1991.

7. Noakes DE, Parkinson TJ, England GCW: Arthur’s Veterinary Reproduction and Obstetrics, 8^th ed. Philadelphia, WB Saunders, 2001, pp. 3, 4, 33.

8. Olsen et al: Vaginal Cytology. Part I. A useful tool for staging the canine estrous cycle, Compendium for the Continuing Education for Practicing Veterinarians, 6:288-289, 1984.

9. Olson et al: Vaginal Cytology. Part II. It’s Use in Diagnosing Canine Johnson, C.A.: Diagnosis and Treatment of Chronic Vaginitis in the Bitch. Reproductive Disorders. Compendium for the Continuing Education of Practicing Veterinarians. 6:385-390; 1984.

10. Raskin RE, Meyer DJ: Atlas of Canine and Feline Cytology. Philadelphia, WB Saunders Co, 2001, pp. 277-312.

11.Rogers KS: Transmissible venereal tumor. Compend Contin Educ Pract Vet 19:1036-1044, 1997.

12. Thrall MA, Olson PN: The vagina. In: Cowell RL, Tyler RD, Meinkoth JH: Diagnostic Cytology and Hematology of the Dog and Cat, 2^nd ed. St. Louis, Mosby, Inc., 1999, pp. 240-248. (from reference #2)

Acknowledgement

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