cuff is subsequently deflated and the catheter removed from the reproductive tract.
An alternative method involves passage of an insemination pipette into the uterus and the infusion of 60 ml of 0.9% sodium chloride via a syringe. The uterus is massaged to distribute the fluid and then the fluid is aspirated back into the syringe. Fluid recovery may be more difficult with this technique.
Recovered fluid is placed into one or two 50 ml centrifuge tube(s) and centrifuged at 400× g for 10 minutes. Alternatively, the cellular material may be allowed to settle by gravity for a few hours.
All but 5 ml of supernatant is removed and the pellet is re‐suspended in the 5 ml of lavage fluid.
Sterile culture swabs may be placed into the re‐suspended lavage fluid to obtain samples for cytology and culture. The culture swab is transferred to a transport media and the cytology swab is gently rolled across a microscope slide. The slide is air‐dried prior to staining with a modified Wright’s stain such as Diff‐Quik®.
An additional sample may be obtained from the centrifuge tube for polymerase chain reaction (PCR) analysis for bacterial or fungal DNA.
Interpretation
It is important that sufficient fluid is recovered from the uterus to be of diagnostic use.
Enough fluid should pass through the lavage tubing so that assessment of the uterine environment is performed. Care should be taken to not just pass fluid into and out of the catheter without actually contacting the uterus.
Note any discharge present on the sterile sleeve when the gloved arm is pulled from the vagina. There should not be any purulent or hemorrhagic discharge, only the lubricant and clear mucous should be present.
The clarity and presence of debris and mucus should be noted in the effluent fluid recovered from the uterus. Typically, the recovered fluid is graded as clear, cloudy, or cloudy with mucus.
Microscopic examination is performed at 400× (10× eye piece and 40× objective) magnification or under oil immersion at 1,000× (10× eye piece and 100× objective).
Evaluate slide quality (e.g., the presence of normal uterine epithelial cells) as well as the presence and relative number of inflammatory cells (neutrophils, macrophages, lymphocytes, etc.) and any bacterial or fungal organisms.
The amount of background debris and sediment appearing on stained slides from a low volume lavage is often greater than with swab or brush techniques. The amount of debris may be correlated with the degree of inflammation.
The presence of >5–10 neutrophils per high power field is an indication of inflammation, providing that an adequate cellular sample was obtained.
The greater the ratio of neutrophils : uterine epithelial cells, the greater the degree of inflammation or endometritis.
Neutrophils are the primary white blood cells present with acute inflammation. Macrophages, lymphocytes, and plasma cells may be noted with chronic inflammation. Eosinophils may be noted in cases of fungal endometritis, urine pooling, or aspiration of air into the uterus.
The absence of white blood cells (with normal endometrial cells present) often suggests that there is no inflammation present in the uterine lumen. However, some bacteria such as Escherichia coli and Pseudomonas aeruginosa may not stimulate a large inflammatory response and a “negative cytology” (i.e., the absence of white blood cells) may provide incorrect evidence of a “clean” or non‐infected uterus. Consequently, it is recommended that both culture and cytology samples be collected and evaluated and not just one or the other.
Please see Chapter 17 for further description of cell types found on cytology.
Further Reading
1 Ball BA, Shin SJ, Patten VH, Lein DH, Woods GL. 1988. Use of a low‐volume uterine flush for microbiologic and cytologic examination of the mare’s endometrium. Theriogenology 29: 1269–83.
2 Brook D. 1993. Uterine cytology. In: McKinnon AO, Voss JL (eds). Equine Reproduction. Philadelphia: Lea and Febiger, pp. 246–54.
3 Couto MS, Hughes JP. 1984. Technique and interpretation of cervical and endometrial cytology in the mare. J Eq Vet Sci 4: 265–73.
4 Leblanc MM, Magsig J, Stromberg AJ. 2007. Use of low‐volume uterine flush for diagnosing endometritis in chronically infertile mares. Theriogenology 68: 403–12.
19 Endometrial Biopsy
Patrick M. McCue
Equine Reproduction Laboratory, Colorado State University, USA
Introduction
Biopsy of the uterine lining or endometrium is primarily used in the evaluation of uterine health, detection of uterine disease, and as a prognostic indicator of the ability of a mare to carry a foal to term. Endometrial biopsies are collected as part of a routine breeding soundness evaluation, as part of a pre‐purchase evaluation of a potential broodmare, and in the examination of barren mares, problem breeding mares, and mares with a history of pregnancy loss. Samples may be collected prior to the onset of the breeding season, during the breeding season, or at the end of the breeding season. A biopsy may be collected during any stage of the estrous cycle, although individual veterinarians may prefer to collect samples when the mare is either in diestrus or in early estrus. It is important to record the stage of the mare’s estrous cycle so that the pathologist evaluating the biopsy sample can interpret the architecture accordingly.
Endometrial biopsy samples may also be used for microbiological culture, cytologic evaluation, and detection of microbial DNA using reverse transcription polymerase chain reaction (RT‐PCR) techniques.
Equipment and Supplies
Uterine biopsy instrument, sterile obstetrical sleeve, sterile obstetrical lubricant, formalin (10%) or Bouin’s solution, alcohol (70%), 25 gauge needle to tease biopsy out of instrument.
Technique
An ultrasound examination is recommended prior to biopsy collection to confirm that the mare is not pregnant and to help stage the mare’s estrous cycle.
The tail of the mare is wrapped and held out of the way (see Chapter 4).
The perineal area is thoroughly cleansed using a non‐residual liquid soap, rinsed with clean water, and dried with paper towels (see Chapter 3).
A sterile obstetrical sleeve with sterile lubricant is worn by the examiner.
An endometrial biopsy instrument is held in a closed position within the hand, guided into the vagina, and then carefully passed through the cervix
