Diagnostic Value of Transcervical Endometrial Biopsies in Domestic Dogs Compared with Full-Thickness Uterine Sections

Authors


Author's address (for correspondence): BW Christensen, Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA 50011, USA. E-mail: drbruce@iastate.edu

Contents

Transcervical endometrial biopsy is a useful tool for obtaining information about uterine health in some species. The clinical application of information gained from histopathological interpretations of endometrial biopsies in the bitch has not been validated. We hypothesized that transcervical endometrial biopsy samples would be as diagnostic as full-thickness uterine sections in identifying cystic endometrial hyperplasia (CEH), inflammation and periglandular fibrosis. Endometrial biopsies were obtained from 20 female adult dogs. Vaginal swabs, gross appearance of the vulva and vaginal tract, and serum progesterone values were used to determine the stage of the oestrous cycle at the time of sampling. The uteri were removed between 1 and 6 days after the biopsy procedure, and full-thickness sections were collected from each uterine horn and ovary and processed for histopathology. Two pathologists, blinded to the origin of each sample, compared full-thickness sections from the excised uteri to the biopsy samples collected via the transcervical technique. Pathologic features noted included: CEH, inflammation and periglandular fibrosis. Pathological diagnoses obtained from the biopsy sections were compared with those obtained from the full-thickness sections, as well as comparing diagnoses between the two pathologists, using McNemar's test. Of the 59 total biopsy samples obtained, 54 were considered diagnostic. All stages of the canine oestrous cycle were represented (anoestrus, proestrus, oestrus and dioestrus). Pyometra was not noted in any of the transcervical biopsy sections, but was noted in many of the full-thickness sections collected from dogs in dioestrus, suggesting either that biopsy is not a sensitive indicator of pyometra or that the procedure may induce pyometra in dioestrous dogs. Transcervical endometrial biopsy showed similar sensitivity as full-thickness sections in detecting CEH, inflammation and fibrosis. No differences in describing lesions were detected between pathologists.

Introduction

Cystic endometrial hyperplasia (CEH) is a common condition in female canids and predisposes affected animals to develop pyometra, a life-threatening condition (Schlafer and Gifford 2008). Recently, a number of captive, wild canids have been diagnosed with CEH and pyometra, resulting in spontaneous death or euthanasia (Boutelle and Bertschinger 2010; Jankowski et al. 2012). These cases underscore a need for a diagnostic tool to identify animals at risk before the disease becomes life threatening. Such a tool may also be used in domestic dogs to determine causes of infertility in breeding bitches. Ultrasound has been shown to be a valuable tool in experienced hands for diagnosing advanced CEH (Bigliardi et al. 2004), but is not useful for diagnosing other pathological conditions, such as endometritis or early cases of CEH. Transcervical endometrial biopsy is useful in diagnosing a number of endometrial pathological conditions in other species, but has been reported as not effective in the bitch (Günzel-Apel et al. 2001). The purpose of this study was to compare the pathological findings in transcervical endometrial biopsies from the domestic bitch to pathological findings in full-thickness sections from the same uterus.

Materials and Methods

Vaginal swabs for cytological samples and 3 ml of blood for serum progesterone assay were obtained from 20 adult, intact, female domestic dogs at various stages of the oestrous cycle. None of the animals had obvious clinical signs consistent with any underlying disease. Nine dogs had known birthdays and ranged in age from 5 to 8 years old. Breeds of dog (weights included in kg) represented were Basset hound (20.8), Bichon frise (6.8), Beagle (5.9, 6.8, 7.3, 7.8), Boxer (18.7), Cairn terrier (5.9), English coonhound (13.6), German shepherd dog (22.3), Lhasa apso (6.5, 7.6, 8.2), Schnauzer (6.7, 6.8, 8.3), Shar pei (18.5), Shih tzu (6.0, 6.2) and Staffordshire terrier (29.0). Vaginal cytologies were interpreted in accordance with accepted clinical practice, using the presence and number of red blood cells, neutrophils and types of epithelial cells (parabasal, intermediate and superficial) to estimate the stage of the oestrous cycle (von Heimendahl and England 2010). Turgidity of the vulva of each bitch was noted; a swollen, turgid vulva was considered a positive sign of proestrus (secondary to elevated serum oestrogen concentrations).

Blood samples were centrifuged, and serum was frozen at −20°C until evaluation. Dog serum samples were analysed using radioimmunoassay (RIA) in the Endocrinology Laboratory at the Saint Louis Zoo. Progesterone was measured using a commercially available RIA kit (Coat-A-Count© Progesterone 125I Kit; Diagnostic Products Corp, Los Angeles, CA, USA). The standard curve ranged from 0.95 to 263.94 nm, and the sensitivity of the assay is 0.06 nm. In total, one assay was performed. The assay was run according to kit directions, with the exception that the progesterone kit standards, which are supplied in human serum, were replaced by standards obtained from Sigma Chemical (Saint Louis, MO, USA) and diluted in a 10% steroid-free calf serum/90% phosphate-buffered saline solution. These standard concentrations were within the range of those provided by the manufacturer and above the minimum sensitivity of the assay. While specific binding did not differ in the presence of 10% steroid-free calf serum and 0 standard provided by the manufacturer, specific binding was lower in the presence of steroid-free dog serum. To account for this difference and equalize the matrix between standard and sample tubes, 10% steroid-free calf serum was added to the dog serum samples, and steroid-free dog serum was added to the standards and quality controls. Dog and calf serum were stripped of steroids using dextran-coated charcoal (DCC# 6241; Sigma Chemical, Saint Louis, MO, USA) prior to use. All samples were assayed in duplicate. Mean ± SE intra-assay variation of duplicate samples was 6.47 ± 0.28%. Serum progesterone values <3.18 nm were considered baseline and consistent with anoestrus; values >3.18 nm were considered consistent with luteal activity and correlated with proestrus, oestrus or dioestrus.

Each dog was restrained manually for the transcervical biopsy procedure. If an individual animal required more than minimal manual restraint to stand still, an intravenous injection of xylazine (0.2 mg/kg), butorphanol (0.2 mg/kg) and glycopyrrolate (5 μg/kg) was administered. A rigid, 43 cm endoscope with a five French instrument port was passed transvaginally in each dog, and the appearance of the vaginal mucosa was noted. Development of vaginal mucosal oedema was considered a positive sign of elevated serum oestrogen concentrations, consistent with proestrus (von Heimendahl and England 2010). Upon visualization of the cervix, a five French biopsy instrument was passed transcervically into the uterus. Three separate biopsy samples were taken from 19 of the bitches and two samples from one of the bitches (due to difficulty restraining the animal). Between each sample, the endoscope was removed from the vagina prior to retrieval of the tissue sample. The endoscope was cleaned and sanitized between each bitch, but not between samples from the same bitch.

Each tissue sample was placed in 4% paraformaldehyde for 24 h and then in phosphate-buffered saline for 24 h; samples were subsequently stored in 30%, 50% and 70% ethyl alcohol for 24 h each prior to further processing for histopathological evaluation. Sections were placed in paraffin blocks and then deparaffinized and hydrated to water by passing the sections through xylene (two changes, 3 min each), absolute ethanol (two changes, 1 min each), 95% ethanol (1 min) and 70% ethanol (1 min). Sections were rinsed in distilled water for 1 min, stained in ‘Shandon Instant Haematoxylin’ for 5 min, and rinsed in tap water for 1 min. Sections were then dipped twice in 0.5% acid alcohol to remove background haematoxylin and differentiate nuclear staining, followed by a rinse in tap water for 24 s. Sections were then placed in blue ‘Scott's Tap Water Substitute’ for 1 min and then rinsed in tap water for 24 s. Sections were then dehydrated in 80% ethanol for 1 min. Sections were then stained in Eosin solution in 95% ethanol for 30 s and then the Eosin was differentiated by placing the sections in 95% ethanol for 30 s. Sections were rehydrated and cleared by placing them in absolute ethanol (three changes, 30 s each) and xylene (three changes, 1 min each). The sections were then placed beneath a coverslip.

Between 1 and 6 days later each bitch was euthanized, the reproductive tract grossly evaluated and a full-thickness section from midway along each uterine horn was retrieved. Euthanasia was performed because these dogs were part of another project that required euthanasia. The other project did not involve the reproductive system, nor did it affect the systemic health of the dogs. The variable days chosen for euthanasia in relation to the biopsy sampling were necessary due to availability of the dogs in the primary project. Samples were fixed in paraformaldehyde and processed as described previously for the biopsy sections.

Two pathologists, who were blinded as to the origin of each sample, independently evaluated each biopsy and full-thickness sample (Figs 1-4). Uterine and ovarian sections were evaluated to determine the stage of the oestrous cycle by assessing endometrial surface epithelial height and vacuolation, polarity of the epithelial nuclei, presence of endometrial haemorrhage and hemosiderin and degree of oedema. When surface epithelium was not present in a biopsy, no assessment could be made. In full-thickness sections, the ipsilateral ovary was evaluated for the presence of primary, secondary, tertiary and Graafian follicles as well as the presence and quality of corpora lutea. As vaginal cytology studies or serum progesterone assays from one single day may demonstrate similar appearances between bitches in proestrus and in dioestrus, histological evaluation was considered the definitive observation for the diagnosis of the stage of the oestrous cycle.

Figure 1.

Full-thickness uterus, canine #2. Note the mild lymphoplasmacytic inflammatory infiltrate in the endometrium. Inset depicts a transcervical biopsy specimen, demonstrates the relative size of the sample and also shows a mild lymphoplasmacytic inflammatory infiltrate in the superficial endometrium, intact luteal epithelium, myometrium and glandular distention in deep endometrium. Haematoxylin and eosin (HE). 2 × magnification

Figure 2.

Full-thickness uterus, canine #6. The endometrium has increased cellularity and some luminal debris and inflammatory cells (arrowhead). This arrowhead identifies the area magnified in Fig. 3. Haematoxylin and eosin (HE). 2 × magnification

Figure 3.

Full-thickness uterus, canine #6, higher magnification of Fig. 2. Luminal suppurative exudates and debris (large arrowhead) as well as tall vacuolated luteal epithelium (thin arrow) are evident. There is also markedly increased number of lymphocytes and plasma cells in the superficial endometrium (thicker arrow). Haematoxylin and eosin (HE). 20 × magnification

Figure 4.

Transcervical biopsy of uterus, canine #6. Note the large dilated cystic gland in the superficial endometrium filled with inspissated eosinophilic contents (arrowhead). The epithelium overlying this papillary projection of endometrium is tall vacuolated luteal epithelium, and there are minimal adjacent lymphoplasmacytic infiltrates. Haematoxylin and eosin (HE). 10 × magnification

Each uterine section was evaluated for presence or absence of inflammation, CEH and fibrosis. McNemar's test was performed to detect the differences in positive diagnoses between biopsied samples and full-thickness samples for each pathological finding, as well as differences in interpretation between the two pathologists. Presence or absence of pyometra was also noted for each of the histopathological sections. The Institutional Animal Care and Use Committee at Iowa State University approved this trial.

Results

Diagnostic results were obtained from 54 of 59 biopsy samples (91.5%). Many biopsy samples went deep into the tissues and recovered basal glands and myometrium. Three dogs were determined to most likely be in anoestrus, eight in proestrus, one in oestrus and eight in dioestrus (two in metoestrus, six in mid- to late dioestrus).

At least one pathologist diagnosed cellular infiltrates in at least one of the samples from 90% (18/20) of the dogs in this study (Table 1), 50% of dogs were similarly diagnosed with endometritis, 35% of dogs with CEH (7/20) and 30% with fibrosis (6/20).

Table 1. Biopsy to spay: indicates the number of days between the transcervical biopsy procedure and removal of the uterus for full-thickness section collection
Dog #Biopsy to spay (days)P4 (ng/ml)Oestrous cycle stagePyoCIEMCEHFib
  1. P4: serum progesterone concentration on the day of transcervical biopsy; Oestrous stage: P, proestrus; E, oestrus; M, metoestrus (early dioestrus); D, dioestrus (mid- to late); A, anoestrus; for the remaining columns (Pyo, pyometra; CI, cellular infiltrates; EM, endometritis; CEH, cystic endometrial hyperplasia and Fib, fibrosis), presence is indicated with a ‘Y’ if the lesion was noted by either pathologist in any of the tissue samples from that animal.

14<0.33PNYNNY
243.36DYYYYN
3418.23DYYYYY
440.49DNYYNN
541.14DYYYYY
644.52DYYYYN
740.40ANYNNN
83<0.33ANYYNY
94 N/AANYYNN
1057.65DYYYYY
1114.32ENNNNN
1211.07DNYYYY
135<0.33PNYNNN
1410.56PNYYYN
1560.85PNYNNN
165 N/APNYNNN
1711.74PNYNNN
18548.46MNNNNN
195<0.33PNYNNN
206<0.33PNYNNN

Pyometra was not detected in any of the biopsy tissue sections, nor on any of the full-thickness sections of dogs in anoestrus, proestrus or oestrus. Pyometra was, however, detected on the full-thickness sections in five of the eight dogs biopsied in mid- to late dioestrus (Figs 2 and 3), all of which had full-thickness sections harvested 4 or 5 days after the biopsy procedure. Each of these five animals, on the day of retrieval of the uterus, was noted to have a mucopurulent vulvar discharge. All five of the dogs with pyometra had evidence of concurrent CEH and endometritis on full-thickness sections, three of which had evidence of CEH and two with endometritis in biopsy samples, as well. Of the three dogs biopsied in dioestrus that did not develop pyometra by the time the uterus was harvested, one had concurrent CEH diagnosed on both biopsied and full-thickness sections; the other two had no evidence of CEH. Of these three dogs, one was estimated to be in metoestrus, one in mid- to late dioestrus and one was estimated to be in late dioestrus. The uterus from one of these latter two dogs had to be harvested 1 day after the biopsy was taken; the other uteri from the remaining dogs in dioestrus were harvested 4–5 days after the biopsy procedure.

No differences were detected between the two pathologists in the consistency of detecting any of the lesions studied (p > 0.05). The difference in proportion of positive results between pathologist A and pathologist B was 5% for diagnosing inflammation using biopsy samples (75% and 70%, respectively; p = 1.00), 0% for diagnosing inflammation using full-thickness samples (80% and 80%, respectively; p = 1.00), 5% for diagnosing CEH using biopsy samples (15% and 20%, respectively; p = 1.00), 10% for diagnosing CEH using full-thickness samples (20% and 30%, respectively; p = 0.50), 10% for diagnosing fibrosis using biopsy samples (10% and 0%, respectively; p = 0.50) and 10% for diagnosing fibrosis using full-thickness samples (15% and 5%, respectively; p = 0.625).

There was no evidence of a difference between biopsy samples and full-thickness samples for the occurrence of cellular infiltrates, endometritis, CEH or fibrosis. The difference in proportion of positive results between transcervical biopsy and full thickness sections was 5% for cellular infiltrates (75% and 80%, respectively; p = 1.00), 15% for endometritis (25% and 40%, respectively; p = 0.45), 5% for CEH (15% and 20%, respectively; p = 1.00) and 5% for fibrosis (10% and 15%, respectively; p = 1.00).

Discussion

The high percentage of diagnostic samples obtained in this trial (91.5%) using transcervical endometrial biopsy is in contrast to a previously reported trial in which only 31.1% of biopsies were considered diagnostic and no basal glands or myometrium were observed (Günzel-Apel et al. 2001). Because diagnostic samples can be reliably obtained, and there is strong agreement between pathologists in interpretation of the samples, transcervical endometrial biopsy may be regarded as reliable a method as evaluating full-thickness uterine sections for detecting inflammation, CEH and fibrosis.

The high rate of inflammation diagnosed in this study (90% of dogs showed some degree of inflammation) may not be clinically important as many of the cases (40%) were deemed to exhibit mild changes with only some cellular infiltrates. Even so, 50% of dogs exhibited moderate (20%) to severe (30%) inflammatory changes and were thus classified as having endometritis (Table 1). This surprisingly high percentage of dogs with endometritis was unexpected and more work would need to be performed to determine how representative this is in the general population.

Given the high mortality that accompanies CEH and endometritis, this technique may be used as a valuable screening tool to detect animals in the early stages of disease when treatment may better help avoid the life-threatening clinical signs of more chronic disease. Pyometra, which is usually associated with both CEH and endometritis, is a disease of high concern for owners of older, intact, nulliparous domestic bitches. Pyometra has also been shown to be a deadly condition in older, multiparous African hunting dogs (Lycaon pictus) with a recent history of chemical contraception with leuprolide acetate (Jankowski et al. 2012).

The variable number of days (from one to six) between transcervical biopsy and uterine retrieval for full-thickness sections was an unavoidable source of variation in this study, due to the fact that these animals were also part of another study and were only available for biopsy on specific days. In assessing potential risks associated with this technique, it would be better to have a standardized number of days between the transcervical biopsy and the full-thickness sections. In addition, there is a question of how representative each of these samples may be to the uterus in general. In other words, how diffuse are these lesions? Future work should focus on these questions.

The high rate of pyometra in this study in mid- to late dioestrous bitches detected in full-thickness uterine samples taken 4–5 days after transcervical endometrial biopsies, but not detected in the transcervical biopsies, themselves, suggests that either bitches in dioestrus are predisposed to development of pyometra following this procedure or possibly that transcervical endometrial biopsies may not effectively detect pyometra. Mechanical disturbance has been shown to effectively induce CEH in bitches in early to mid-dioestrus (Nomura et al. 1990; Nomura 1997; Verstegen et al. 2008), compared with late dioestrus, using behaviour and serial vaginal cytology to determine the stage of the cycle. In the current study, four of five pyometra cases seemed to develop in bitches biopsied in late dioestrus (based on histology and serum progesterone concentrations). In addition, one of the three dioestrous cases that did not develop pyometra was biopsied in early dioestrus. It would seem based on the previously cited work and the findings of this study that it would be best to avoid the luteal phase altogether when performing biopsy procedures.

Introduction of E. coli into the dioestrous uterus of the bitch has also been shown to induce pyometra (Nomura and Funahashi 1999). Iatrogenic introduction of bacteria into the uterus using the technique described in this study is a possibility as three endometrial samples were desired from each animal, and it was not considered practical to sterilize the instruments prior to obtaining the second and third samples, although every effort was made to keep the instruments from touching anything besides the internal reproductive tract of the bitch. Taking the time necessary to sterilize the endoscope and biopsy instrument between samples would have been impractical. Modifying the technique to only obtain one endometrial sample instead of three, while reducing the chance for introducing bacteria into the uterus, would markedly reduce the sensitivity of this technique to detect CEH as never in this study did either pathologist diagnose CEH on all three transcervical endometrial samples from the same uterus. In fact, in two of the five cases diagnosed with CEH using transcervical endometrial biopsy, only one of the three tissue samples taken exhibited evidence of CEH. Until these possibilities are examined more carefully, caution is warranted when performing transcervical endometrial biopsy in the bitch to avoid the dioestrous period. It is recommended that diagnostic tests such as serial serum progesterone concentration assays and vaginal cytology be performed to avoid dioestrus when performing transcervical procedures.

Acknowledgements

The authors wish to thank the Saint Louis Zoo for funding. In addition, thanks to Dr Larry Evans, Dr Lin Kauffman, Ms Rochelle Panthen, Dr Jamie Petersen, Dr Stacey Hoffman, Dr Sarah Tully, Dr Julie Wilfong, Dr Vicki Riveland, and Dr Shawn Thomas for technical support with the dogs.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

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