Luisa Ramírez‐Agámez1, Camilo Hernández‐Avilés2, and Chelsea Makloski‐Cohorn3 1 Animal Reproductive Services, Bogotá, Colombia 2 Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA 3 Pinnacle Equine Veterinary Services, PLLC, Whitesboro, TX, USA As with the stallion, the evaluation of a mare’s reproductive health is based on the assessment of breeding records, physical examination of the reproductive tract and the use of complementary tests to determine subclinical or clinical conditions that cannot be obviously diagnosed during the physical exam. Although a complete description of the breeding soundness examination of the mare is beyond the scope of this chapter, it is important to remember that the examination of the mare should include [1]: In general terms, the use of endometrial cytology is less common amongst veterinary practitioners compared to the use of endometrial culture, particularly in busy private practices. This is due to inconsistent results, lack of familiarity with the technique or difficulty in the evaluation and interpretation of the cell types present [2]. However, the use of two or more diagnostic aids when performing a thorough reproductive examination is necessary to adequately determine whether or not a mare is suffering from an endometrial inflammatory process (endometritis), and to determine the course of action that should be taken [1–4]. In fact, some studies have demonstrated that the use of endometrial cytology in conjunction with endometrial culture or biopsy is related to a higher capacity to accurately detect endometritis in subfertile mares, particularly when this condition is related to the presence of bacteria such as Streptococcus zooepidemicus, Staphylococcus aureus or Klebsiella pneumoniae [2, 5, 6]. Thus, it is imperative that veterinarians become familiar with the adequate collection, processing, and interpretation of endometrial cytology. This chapter will focus on the most common techniques for collecting and processing samples for cytological analysis and their interpretation. Since their introduction in the early 1960s, several techniques have been reported for the collection of cytological specimens. Most of these sampling techniques have included the use of uterine culture swabs, double‐guarded culture swabs, uterine cytology brushes, uterine biopsies, or specimens resulting from uterine lavage fluids. Although all these techniques have their potential advantages and disadvantages, practitioners must be aware that regardless of the technique chosen for sampling, these must (i) be harmless to the mare’s endometrium, (ii) avoid the introduction of pathogens to the uterine environment, and (iii) facilitate the collection of sufficient numbers of endometrial and other cells to allow a reliable cytological examination. The decision to use one technique or another should also depend on the preferences of the practitioner in terms of ease, associated costs, and time required. The acquisition of cytological samples is most commonly done during midestrus, since at this stage of the cycle the cervix is open and the uterine immune mechanisms are more active [7]. Hence, the likelihood of finding inflammatory cells (neutrophils) or bacteria will be increased [8, 9]. Others have suggested that endometrial cytology should be acquired during late diestrus–early estrus, particularly if that cycle is intended to be used for breeding the mare [8]. However, the odds of producing more false‐negative results, as well as the chances of inducing an iatrogenic infection of the endometrium, are higher when diestral cytology is taken [9, 10]. Prior to sampling, the mare should be placed in stocks and prepared in the same way as for artificial insemination. This implies that all feces must be removed from the rectum, and the vulva, perineum, and perianal region must be scrubbed using either a chlorhexidine‐ or iodine‐based solution and washed with clean water. Particular care must be taken when cleaning the vulvar labia and clitoral fossa, since these places harbor several commensal and potentially pathogenic bacteria that could be accidentally infused into the mare’s endometrium [11]. The practitioner must wear a sterile sleeve and place the tip of the culture device (swab or uterine catheter) into the palm of the hand, to avoid it coming into contact with lubricant, the skin or mucosa whilst passing through the vulva and vagina. Sterile, nonbacteriostatic lubricant should be placed on the top of the hand, making sure that the tip of the culture device is not contaminated with the lubricant. Then, the hand and the culture device are passed through the vulvar labia, vestibule, and vagina until reaching the external cervical os. Subsequently, a finger is used to guide the culture device through the cervical os and then into the uterine lumen. At least three different types of culture swabs have been commonly described for cytological examination of the mare’s endometrium. The Kalayjian single‐guarded uterine swab (Kalayjian Industries Inc., Long Beach, CA) (Figure 16.1) and the McCullough double‐guarded uterine swab (McCullough Cartwright, Barrington, IL) (Figure 16.2) are the most commonly used for this purpose, with the latter mostly preferred by the authors [2, 3, 12–14]. All of them comprise outer guarded tubes with an inner rod holding a calcium alginate swab. Calcium alginate swabs are better than cotton swabs due to the likelihood of having cotton fibers on the stained smear which may interfere with the staining process or cytology interpretation [12]. The use of double‐guarded swabs is more advantageous in terms of avoiding sample contamination with cells and bacteria from the mare’s vagina or cervix [13]. Figure 16.1 The Kalayjian single‐guarded endometrial swab. Observe the cap in the outer plastic tube, which is used to collect endometrial cell samples. Figure 16.2 The McCullough double‐guarded endometrial swab. (Top) Appearance of the double‐guarded swab prior to passing it through the mare’s reproductive tract. (Middle) The inner plastic tube is propelled through the tip of the culture device. (Bottom) The swab is propelled through the inner plastic tube to take the sample from the uterine lumen. After passing the swab through the cervix, the swab is pushed forward from its plastic guard. It is recommended to roll the swab against the endometrium and allow contact between the swab and the uterus for at least 30 seconds (for microbiological analysis). Then, the swab is retracted back into the plastic guard and removed from the reproductive tract, in order to avoid sample contamination. Recently, the use of cytological brushes (Minitube GmbH, Tiefenbach, Germany) (Figure 16.3) has become more popular for obtaining samples for cytological analysis, particularly in North America and Europe. The main advantage of using a cytological brush over a culture swab is the capacity to obtain a high proportion of diagnostic smears, with more cellular intactness and less chance of preparation artefacts [15–17] (Figure 16.4). The procedure for taking samples for endometrial cytology is the same as for guarded swabs. Practitioners may also prefer to collect a sample for endometrial cytology by passing the culture swab or cytological brush through a sterile disposable vaginal speculum (Figure 16.5). Using this procedure is advantageous in mares with vulvoplasty (Caslick), to avoid inconvenience whilst passing the hand through the vulvar labia. Also, this technique has been described to reduce the incidence of sample contamination for microbiological analysis when compared to passage of the guarded swab using a sterile sleeve and the hand [18, 19]. Figure 16.3 A cytological brush (Cytobrush). The system used to protect the brush from contamination is the same as for the double‐guarded endometrial swab. Figure 16.4 Jackson uterine biopsy forceps without (left) and with (right) sample. Cytological samples obtained from endometrial biopsies are more cellular. Figure 16.5 Passing an endometrial culture device through a speculum. This method is helpful in mares with Caslick suture as well as to prevent some degree of contamination, particularly when the endometrial swab is passed through the vulva and vagina. Although both methods described above are easy to conduct and inexpensive under field conditions, disadvantages of their use are related to the fact that the samples obtained may not be completely representative of the entire endometrial surface; thus, the odds of misdiagnosing endometrial inflammation are increased. Several studies have demonstrated that the use of only guarded swabs for diagnosis of endometritis is not sufficient and may lead to false‐negative results, when compared to the use of two or more diagnostic techniques, or when compared to other techniques for sample acquisition (uterine lavage or biopsy) [4, 6, 15, 17]. In the same way, it is always recommended to take samples for uterine cytology and culture, since the capacity to truly detect endometritis, particularly infectious endometritis due to Escherichia coli or Pseudomonas aeruginosa, is improved [3, 20]. The initial descriptions of the use of uterine lavage for recovering material intended for endometrial cytology were published in the mid‐late 1980s. Ball et al. demonstrated that the infusion of 60 mL of phosphate‐buffered saline (PBS) solution into the uterine body and its subsequent recovery improved the capacity to obtain microbiological and cytological samples from subfertile mares [21]. Recent studies have suggested that the use of uterine lavage is advantageous in correctly diagnosing mares that suffer from endometritis, compared to the use of swabbing techniques alone [6, 16, 17, 22]. In most of these reports, the infusion of approximately 50–60 mL of a physiological solution (PBS, lactated Ringer’s or saline solution) was performed; however, the recovery rate of fluid by using this method is relatively low (30–35 mL on average). Even when this method is superior to the use of swabs for sampling a wide area of the uterus, some authors believe that the volume infused is not enough to cover the whole surface of the endometrium [23]. Thus, other researchers have used up to 250 mL of fluid to recover endometrial cells for cytological and microbiological analysis with good results [16, 24]. In the authors’ opinion, the use of 250 mL or up to 3–6 L of fluids for lavage may have not only a diagnostic but also a therapeutic benefit, particularly in mares suffering postbreeding endometritis [25]. We consider that the infusion and lavage of the uterus with large volumes of fluid may help to reduce the inflammatory response, as has been previously demonstrated, as well as helping to remove cellular debris and bacteria present in the uterus [25, 26]. After recovering the fluid from the uterus, the samples can be collected by centrifugation (200–600 g × 10 minutes) and then smeared and stained for analysis. In order to perform a low‐volume uterine lavage for endometrial cytology, the practitioner has to clean the mare’s perineal region, as described above. Then, a sterile uterine catheter is passed through the vagina and cervix, as described above for taking endometrial swabs. Most practitioners use a 60–80 cm uterine lavage silicone catheter with an air cuff, in order to place the tip of the catheter inside the uterus without worrying about losing fluid through the cervix. After passing the catheter, single‐line plastic tubing attached to a 150 mL sterile saline solution is connected to the end of the catheter. A syringe is connected to the cuff of the catheter and inflated with approximately 75 mL of air. Then, the cuff should be gently pulled out to verify that the catheter is correctly placed against the internal cervical os. Finally, the bag containing the sterile saline is elevated, in order to allow the flow of liquid to the uterus by gravity. The fluid is allowed to stay into the uterus for 2–3 minutes. In the meantime, the uterus may be gently massaged transrectally to ensure wide distribution of the fluid. The bag is then lowered (generally to ground level) to allow the return of the fluid by gravity (Figure 16.6). Under some circumstances, particularly in multiparous mares with a pendulous uterus, gently lifting the uterine horns and body can help to recover the infused fluid. Simultaneously, a single IV dose of oxytocin (10–20 IU) can be given to stimulate uterine contractions and increase fluid recovery [27, 28]. As mentioned above, the quantities of fluid that can be used are variable. If larger quantities of fluid are to be used, the authors recommend using either 1 L lactated Ringer’s solution or sterile saline solution connected to the single‐line tubing. Furthermore, a cost‐effective and practical alternative is described by Brinsko [26], in which a gallon of saline solution is instilled into the uterus using a sterile sleeve attached to the end of the uterine catheter. With this method, it is recommended to allow the cells to settle by gravity before processing for cytological analysis.
16
Cytology of the Endometrium
16.1 Sample Collection Techniques
16.1.1 Single‐Guarded and Double‐Guarded Swabs and Cytological Brushes
16.1.2 Uterine Lavage and Low‐Volume Uterine Lavage
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