Why perform a cytology smear in conjunction with a uterine culture?

By Jos Mottershead

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Most responsible stallion owners require a "clean uterine culture" from a mare prior to breeding her live cover or possibly shipping semen to her, and this has been the case for many years. It is well known however, that the results of an endometrial swab culture alone are, at best, not a reliable indicator of uterine pathogenicity, and at worst a false positive may result in disastrous consequences.

A recent Internet poll carried out on the largest non-professional equine reproduction e-mail list¹ indicated that only about 50% of veterinarians routinely perform a cytology smear in conjunction with an endometrial culture. This means that there are still a large number of breeders who are operating under a mistaken belief that their mare is either "clean" when they are not, or who are treating a uterine infection that isn't really there!

What is the significance of the cytology smear?

A microscope slide smear of some of the fluid harvested from the endometrial surface of the uterus is prepared and then stained using a hematology stain. The slide is evaluated for the presence or absence of inflammatory cells, specifically neutrophils.

Normal non-inflammatory endometrial epithelial cells	Inflammatory cells - neutrophils - seen arrowed at left and right with double nuclei
Slide photos courtesy of Dr. Jonathan F Pycock, B.Vet.Med., Ph.D., D.E.S.M., M.R.C.V.S. Equine Reproductive Services, Messenger Farm, Ryton, Yorkshire, England

The combination of endometrial cytology smear and culture results offer the most accurate diagnostic tool connected with uterine swabbing and pathogenicity (an endometrial biopsy is probably more accurate, but is not necessarily cost-effective in all cases). One must consider the various result possibilities:

Culture Growth	Inflammatory Cell Presence	Diagnostic Determination
Negative	Negative	Mare can be considered "clean" and can be bred with no further diagnostic testing required
Positive	Positive	Mare has uterus most likely infected with the pathogen identified in the culture
Positive	Negative	Mare is unlikely to have an infected uterus, and the sample has probably been contaminated (See note on Pseudomonas aeruginosa exception)
Negative	Positive	Mare has something irritating her uterus. In all likelihood, this is a pathogen that swabbing has missed. Further diagnostic work is required

It has been suggested by some that if no neutrophils are identified on a cytology smear, it is not necessary to perform a culture of the sample, and the mare may be considered "clean". It has been indicated to me in personal communication² however, that if Pseudomonas aeruginosa is identified with an absence of neutrophils, this may indicate the presence of the pathogen in a commensal state, which will not develop into an active infection until something triggers the occurrence. That something may be a suppression of the mare's uterine immune system as a result of elevated progesterone levels resulting from pregnancy. It is therefore suggested that a culture should still be completed regardless of absence of neutrophils, although it should be noted that there has not been peer-reviewed research that has proven this to date only clinical evidence, and as this is the only organism that has been suggested to behave in this manner, there is a great likelihood that the absence of neutrophils alone will indicate a "clean" uterus in the vast majority of cases.

If neutrophils are present on the slide, but there is an absence of pathogen growth on the culture, this indicates that there is something irritating the uterus, and it is most likely that it is a pathogen that was missed during sampling. Collection of a surface sample of the endometrial lining of the uterus with the guarded endometrial swab is undoubtedly the easiest method of sampling. Research³ has however suggested that in mares that are considered barren or subfertile, a low volume (60-ml) uterine flush with phosphate buffered saline, introduced via a trans-cervical balloon catheter, in order that the majority of the introduced fluid may subsequently be recovered, yields a more accurate harvesting method to indicate both cytological and bacterial results. A portion of the recovered fluid is concentrated by centrifugation, and after removal of the supernatant and resuspension of the resultant pellet in a lower volume of liquid; a culture is prepared of the harvested matter. The centrifugation is performed to enable a concentrated growth to become apparent, should an organism be present. A cytology smear is prepared using a sample taken from the non-centrifuged portion of the recovered fluid. Preparation using the centrifuged portion would reveal distorted cells that could make interpretation of the results difficult. A practical approach for using this more complicated sampling method rather than the guarded swab, would be to use it only on those mares that have previously proven themselves to be subfertile, or where a previous sample using a guarded swab has resulted in an absence of a pathogen in culture, but the presence of neutrophils in the cytology smear.

Worst case scenario:

In the worst case scenario, a mare with a clean uterus has an endometrial swab performed, which is then subjected to a culture but not a cytology smear. The result is returned as being "positive" for a pathogen, and intra-uterine antibiotic therapy is initiated even though there is actually no pathogen present in the uterus and the diagnosis is erroneous. When one introduces an antibiotic into the uterus, one must consider the possibility of the creation of a "superinfection" where an opportunistic organism - Pseudomonas aeruginosa and yeasts are two such - becomes active. A similar situation is seen in human females who are placed on an antibiotic and subsequently develop a vaginal yeast infection. In the equine, the opportunistic organisms such as those named can be extremely difficult to eradicate, and may lead to permanent damage to the endometrium resulting in alteration of the endometrial biopsy score, and a lowered chance of the mare subsequently producing a live foal.

Summary:

If a cytology smear is not prepared in conjunction with the culture of the swab sample, then the results of the culture as far as showing pathogenic status of the uterus are WORTHLESS!!!! It is essential that if a "positive culture" result is given that there be neutrophils also seen on a cytology smear - without those neutrophils present, it is most likely that the culture result is indicative of a contaminated sample! So - no cytology, no accuracy!

References:

EquineRepro@yahoogroups.com; 1999.
William B. Ley, DVM, MS, Diplomate, ACT; Personal communication, 2000.
Ball, B.A. et. al.; Theriogenology June 1988; "Use of low-volume flush for microbiologic and cytologic examination of the mare's endometrium".

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