Abstract
Objective
To evaluate inflammatory cytokine and chemokine concentrations in endometrial samples collected by cytobrush or swab as a potential screening diagnostic marker for equine endometritis.
Methods
88 mares had endometrial samples collected, which included cytobrush, swab, and/or biopsy. Clinical reproductive records, reproductive ultrasound examination, endometrial cytology and culture results, and biopsy grading score were recorded. Fluorescent bead–based multiplex assays for the inflammatory markers interferon-γ, interferon-α, IL-1β, IL-4, IL-10, IL-17A, soluble CD14, tumor necrosis factor (TNF)-α, C-C motif chemokine ligand (CCL)-2, CCL3, CCL5, and CCL11 were carried out from the endometrial cytobrush and swab samples in reproductively healthy mares and mares with endometritis in a prospective cross-sectional study.
Results
The endometrial cytobrush technique yielded higher inflammatory marker concentrations for mares with endometritis than endometrial swabs. Tumor necrosis factor-α was increased in endometrial cytobrush samples from estrous mares that had endometrial neutrophils on cytology (n = 14; median, 4,185 pg/mL; IQR, 1,318.8 to 7,530 pg/mL) compared to samples from mares without neutrophils on cytology (n = 68; median, 1,653 pg/mL; IQR, 672 to 3,606.8 pg/mL; CI, 288 to 3,715) and in mares with endometritis (n = 14; median, 4,185 pg/mL; IQR, 1,318.8 to 7,530 pg/mL) compared to mares without endometritis (n = 57; median, 1,718 pg/mL; IQR, 651.5 to 3,968 pg/mL; CI, 580 to 4,383). Finally, mares with uterine TNF-α concentrations < 3,942 pg/mL in combination with CCL5 concentrations < 15,985 pg/mL had a 100% chance of identifying as a mare with a healthy endometrium.
Conclusions
Increased concentrations of TNF-α and CCL5 in endometrial cytobrush samples are identified in mares with inflammatory endometritis.
Clinical Relevance
Inflammatory markers measured from endometrial cytobrush samples could be used to identify mares for endometritis that may have false negative results.
Equine endometritis is a costly and frequently encountered disease in clinical practice.1,2 The prevalence of endometritis may account for 25% to 60% of the causes of subfertility of barren mares,3,4 and roughly 28% of clinically normal mares were shown to have subclinical endometritis.5 Screening methods for equine endometritis are commonly used prior to breeding, and they include endometrial cytology and culture. Endometrial cytology and culture, however, lack sensitivity,6–8 and their sensitivity varies from poor to average (17% and 33%, respectively), but if both screening tools are combined, the sensitivity increases to 42%.7 With newer technologies, such as metagenomics, the sensitivity to detect microbes has increased along with the knowledge of the equine endometrial microbiome composition in reproductively healthy mares.9 Nevertheless, routine aerobic cultures are imperative to provide sensitivity results for antimicrobial treatments and guide the veterinarian on the use of appropriate antimicrobials and are unlikely to be replaced by metagenomic technologies.
Endometrial cytology and culture can also be performed from uterine low-volume lavage (LVL), which results in a higher sensitivity than testing performed on endometrial swabs or cytobrushes, but LVL is also more cumbersome as the fluid needs centrifugation, and the cytology is harder to interpret.6,8,10 Hence, LVL is normally reserved for chronically infertile mares.10 Additionally, cutoff values on the percentage or number of neutrophils on cytology are highly variable in the published literature,11 leaving the clinician without firm guidelines on how to classify endometrial inflammation. The gold standard for equine endometritis is the endometrial biopsy as it allows for identification of inflammatory cells deeper in the tissue.6 Additionally, the biopsy grade can be a prognostic indicator of the ability of that mare to carry a foal to term.12 Despite this benefit, a biopsy is invasive and costly and has a longer turnaround time. Hence, it is used more commonly as a breeding soundness evaluation for problematic mares rather than a screening tool prior to breeding. Consequently, fast and reliable screening tools to aid in the detection of mares with endometritis prior to breeding would be an asset to the equine practitioner to improve breeding success.
A group of validated equine-specific inflammatory markers, including C-C motif chemokine ligand (CCL)-2, CCL3, CCL5, and CCL11 along with tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IFN-α, IL-1β, IL-4, IL-10, IL-17A, and soluble CD14 (sCD14), have been shown to be altered in different equine diseases, such as equid alphaherpesvirus-1, foal septicemia, osteoarthritis, and equine asthma13–15 Recently, a pilot study16 has demonstrated that some of these inflammatory markers are increased in mares with acute and chronic endometritis in LVL. In the current study, our aim was to identify specific inflammatory markers in mares with acute inflammatory endometritis via endometrial swab and cytobrush samples as a diagnostic screening tool prior to breeding. Our hypothesis was that the endometrial cytobrush technique would be the preferred sampling method for comparing inflammatory cytokines in mares with and without acute inflammatory endometritis.
Methods
Mares
Healthy mares (n = 88) that were presented at the Cornell University Hospital for Animals or to the Rood and Riddle Equine Hospital for breeding or a breeding soundness evaluation were included in the study. Mares were included if at least 1 of the endometrial samples were collected (endometrial swab and/or cytobrush) for inflammatory markers and if relevant reproductive clinical data were present. Mares’ ages ranged from 3 to 21 years, with a mean of 11.4 years and SD of ± 4.3 years. Besides 3 draft horses and 1 Gypsy Vanner, all of the mares were light-type horses, with the 3 most represented breeds, in order, being Warmblood (26), Thoroughbred (13), and Quarter Horse (9). Excluded from the analysis were mares in anestrus or transition, fillies prior to 3 years of age, and mares with an incomplete reproductive record. Sixteen mares were sampled in diestrus, whereas the remaining 78 were sampled in estrus. Some mares were sampled more than once in different cycles (Supplementary Table S1). All experimental procedures were approved by the Cornell University IACUC (protocol #2019-0102).
Reproductive record
A reproductive examination was performed, and the data collected included age; breed; stage of the cycle; transrectal palpation and ultrasound findings, such as cervical and uterine tone; structures present on the right and left ovaries; presence and grade of endometrial edema (0 to 3, 0 being no edema and 3 being maximal amount of edema); if edema was inappropriate for the stage of the cycle (edema in diestrus or if higher than grade 1 edema present with a smaller-than-30-mm follicle); and presence of intrauterine fluid (yes/no) at the time of sampling. Furthermore, pregnancy status information was collected on the same estrous cycle of the sample collection, between 14 to 25 days after ovulation. Finally, information on whether the mare was susceptible or resistant to postbreeding–induced endometritis was collected based on previous or current breeding records. Mares were classified as susceptible to endometritis if they had fluid accumulation 24 hours after breeding despite uterine lavage. Positive or negative bacterial growth results were also recorded. This study was approved by the Cornell University IACUC (protocol #2019-0102).
Sample collection
In total, 208 samples, 104 endometrial swab and 104 cytobrush samples, were collected for testing in an inflammatory marker assay. Some animals had both an endometrial swab and cytobrush performed, in this order, and some animals had only a single sample. Samples were taken at the time of presentation or in estrus when mares were presented for breeding. Some of the mares were sampled more than once in the same year but always in different cycles. For all mares that had a culture or cytology performed, a separate swab and/or cytobrush were collected for inflammatory marker detection. If samples for bacterial culture were collected, this was always the first sample taken, followed by endometrial cytology, and finally the sample was used for the inflammatory marker assay. Samples were taken via double-guarded swabs or cytobrushes after the mare had her vulva thoroughly cleaned with warm water, clean cotton, and soap. All samples collected for culture or inflammatory markers were placed into a vial with 1 mL of Amies media (Opti-Swab; Puritan). Sixteen mares that presented for a breeding soundness evaluation also had an endometrial biopsy collected for standard histopathological processing and staining with H&E, and histopathological interpretation was performed based on the Kenney-Doig scale12 by a board-certified veterinary anatomic pathologist. The biopsy grading scores were recorded, and the endometrial biopsy was the last sample taken after endometrial cytology and/or culture. The cytology slides were analyzed with a bright-field microscope under the 100X objective with oil immersion for 1,000X magnification. Mares were defined as having endometrial inflammation if there were at least 2 neutrophils/hpf (endometrial epithelial cells and polymorphonucleated cells).17 Endometrial swabs were submitted for bacterial culture to the Animal Health Diagnostic Center at Cornell University or to the Rood and Riddle Equine Hospital Laboratory in Lexington, KY, and data on bacterial growth (positive/negative) were taken. Mares were classified as having endometritis if they had endometrial inflammation on cytology (≥ 2 neutrophils/hpf) regardless of growth on the endometrial bacterial culture. Mares were classified as negative for endometritis if they had fewer than 2 neutrophils/hpf on cytology and had no growth on culture. Mares with positive bacterial growth but without inflammation on endometrial cytology were considered questionable and therefore excluded from the analysis. Fungal endometritis was also excluded from the analysis. A summary of all of the clinical data can be found in Supplementary Table S1.
Fluorescent bead–based multiplex assays for inflammatory markers
The fluorescent bead–based multiplex assays for the quantification of inflammatory markers in horses were performed at the Animal Health Diagnostic Center at Cornell University and have been previously described in detail.13–15,18 Here, we used 3 multiplex assays for cytokines (IFN-α, IFN-γ, IL-4, IL-10, and IL-17A), chemokines (TNF-α, IL-1β, CCL2, CCL3, CCL5, and CCL11), and sCD14 to simultaneously analyze these inflammatory markers in endometrial cytobrush and swab samples. Briefly, different color-coded fluorescent beads (Luminex Corp) were coupled with monoclonal antibodies against each equine inflammatory marker. For each targeted cytokine/chemokine within a multiplex assay, beads of an individual color code were used to distinguish the individual inflammatory markers during measurement of the assay. Immediately before the assay was run, an inflammatory marker standard was prepared using 5-fold dilutions of cytokine/chemokine fusion protein supernatants with known concentrations. MultiScreenHTS plates (Millipore) were used for the assay and were soaked with PBS with Tween 20 buffer using an ELx50 plate vacuum washer (Biotek Instruments Inc) for 2 minutes. After the PBS with Tween 20 buffer was removed, 50 µL of standard curve dilutions or sample was added to the individual plate wells together with 50 µL of bead solution containing 5 X 103 of each bead/well. The plate was covered to protect it from light and incubated for 30 minutes at room temperature on a plate shaker, followed by a washing step. Next, biotinylated monoclonal antibodies for the different equine inflammatory markers were added, and plates were incubated for another 30 minutes as described above. After another washing step, a streptavidin-phycoerythrin solution (Invitrogen), was added, plates were incubated as above and washed afterwards, and the beads were resuspended in blocking buffer. Plates were placed on a shaker for 15 minutes and were then analyzed in a Luminex 200 Instrument System (Luminex Corp). Cytokine and chemokine concentrations were calculated using the standard curves for each inflammatory marker. The data were reported in picograms per milliliter, with the exception of IFN-γ and IL-17A, which were reported in units per milliliter. All sample processing we performed as previously validated.
Statistical analysis
Statistical analysis was performed with JMP Pro (version 17; JMP Statistical Discovery), with the level of significance set at P < .05. The data were evaluated for normality with a Shapiro-Wilk test. A sample size calculation was performed using an expected 15% difference between the 3 groups, and to get an 80% power level at the level of significance of < 0.05, we required at least 76 animals/sampling technique to attain the minimum sample size for the majority of the cytokines of interest. The data in this study were stratified for sampling method by swab and cytobrush to decrease confounding factors as some of the mares were sampled at the same time with both the endometrial swab and cytobrush. The cytokine and chemokine concentrations were compared between the different variables (yes/no inappropriate edema, intrauterine inflammation on cytology, bacterial growth, stage of the cycle, and endometrial biopsy score and yes/no endometritis) with Wilcoxon tests. Due to the small sample size, the biopsy scores were divided into 2 groups: I and IIA or IIB and III. Additional stratification of the data by the stage of the cycle (just mares in estrus or diestrus) for those inflammatory markers that were identified to be statistically different between the stage of the cycle was performed to further decrease bias. The authors have opted for the Wilcoxon statistical approach as a linear regression model would require the data to be normally distributed, and the data were still slightly skewed following log transformation, making this statistical model a better option. Finally, the decision tree predictive modeling, as previously published using an established method,19 was generated from the cytobrush sample data from mares in estrus and used to identify potential inflammatory marker candidate concentrations for detecting mares with endometritis, and a second decision tree analysis was performed to detect an inflammatory marker(s) threshold that could predict pregnancy outcome. The decision tree analysis was used as it is a more robust statistical model to predict the threshold of all inflammatory cytokines to screen mares with endometritis. The decision tree does not assume a linear relationship between responses as a linear regression model would and is able to outperform the linear model by allowing more complicated relationships among variables.20,21
Results
Descriptive statistics
Out of the 104 swabs, 11 were taken in diestrus, and 93 were taken in estrus. Out of the 104 cytobrush samples, 16 were taken in diestrus, and the remaining 88 were taken in estrus. A summary with all of the clinical data can be found in Supplementary Table S1.
Comparison of inflammatory markers in endometrial cytobrush and swab samples
Four out of the 12 inflammatory markers analyzed were below detection limits for most of the samples or at very low concentrations and therefore not included in the analysis: IFN-α was only detected in 1 of 208 samples, IL-4 was detected in 2 of 208 samples, IL-10 was detected in 20 of 208 samples, CCL3 was detected in 15 of 208 samples, IL-17A was detected in 5 of 208 samples, and IFN-γ was detected in 94 of 208 sample. Interleukin-1β was detected in 79 of 208 samples with high concentrations; hence, it was kept in the analysis.
There was no difference in the concentrations of the remaining 6 inflammatory markers in all samples collected between the cytobrush and swab. Figure 1 illustrates the comparison of the inflammatory marker concentration between endometrial cytobrush and swab samples.
Median and SE of the median of inflammatory marker concentrations of C-C motif chemokine ligand (CCL)-2, CCL5, CCL11, tumor necrosis factor (TNF)-α, IL-1ß, and soluble CD14 (sCD14) in picograms per milliliter from endometrial cytobrush (Brush) in gray (n = 104) and Swab samples in red (n = 104). *Difference between groups (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
Inflammatory markers in endometrial swab samples—Endometrial samples from mares with inappropriate edema (n = 7) at the time of sample collection had higher concentrations of CCL5 (P < .01) compared to mares with normal edema (n = 97), but sCD14 was lower in mares with inappropriate edema compared to mares with normal edema for the stage of the cycle (P < .01; Figure 2). In addition, sCD14 was decreased in mares with a positive bacterial culture (n = 33) compared to those with a negative culture (n = 41; P < .01). When examining the effect of cycle, sCD14 concentrations differed between estrus and diestrus (P < .05), and therefore the data were stratified for the stage of the cycle. No difference was found in sCD14 in samples taken in diestrus. In estrus samples, sCD14 was decreased with a positive bacterial culture (P < .05; n = 25) compared to mares that had a negative culture in estrus (n = 39). Supplementary Table S2 shows the summary of the median and IQR for all inflammatory markers and variables analyzed for the endometrial swab samples.
Inflammatory marker concentrations of (A) CCL5 and sCD14 in picograms per milliliter and (B) sCD14 in picograms per milliliter (estrus only; n = 93) detected by fluorescent bead–based multiplex assays in endometrial swabs of mares. Normal edema in gray (n = 97) and inappropriate in red (n = 7), and bacterial culture negative in gray (n = 41) and positive in red (n = 33) in a prospective cross-sectional study. **Difference between groups (P < .03). ***Difference between groups (P < .01).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
Inflammatory markers in endometrial cytobrush samples—C-C motif chemokine ligand 11 was increased in samples of mares with intrauterine fluid detected at the time of sampling (n = 38) compared to mares without fluid (n = 66; P < .05; Figure 3). Additionally, CCL11 was increased in mares classified as susceptible (n = 60) compared to resistant mares (n = 16; P < .05). When mares with intrauterine fluid were removed from the analysis to avoid confounding factors, CCL11 concentrations were not different between susceptible and resistant mares (P < .05). Soluble CD14 was decreased in mares with an inappropriate amount of edema (n = 15; P < .05) compared to those with normal amounts of edema for the stage of the cycle or follicular size (n = 89). There was a difference for TNF-α and sCD14 between estrus and diestrus (P < .05). Hence, the data were stratified between the stage of the cycle. Mares in diestrus with positive cultures (n = 9) had higher concentrations of TNF-α compared to mares with negative cultures (n = 6; P < .01; Figure 4). Mares in estrus had increased concentrations of TNF-α when inflammation was present on cytology (n = 14) compared to samples of mares without fewer than 2 neutrophils/hpf (n = 68; P < .05). Mares with poor scoring (grade IIB and III) endometrial biopsy (n = 2) had much higher concentrations of TNF-α compared to mares with endometrial biopsy grade I (n = 14; P < .05). Finally, mares in estrus classified as having endometritis (n = 14) had increased concentrations of TNF-α compared to estrous mares without endometritis (n = 57; P < .05). Supplementary Table S3 shows the summary of the median and IQR for all of the inflammatory markers and variables analyzed for the endometrial cytobrush. The comparison between inflammatory marker concentrations from endometrial cytobrush samples in estrous mares that were positive or negative for endometritis can be found in Figure 5. Since endometrial cytobrush samples yielded overall higher concentrations of inflammatory markers, as this technique collects from deeper layers of the endometrium compared to the swab, the results from the cytobrush samples were selected for the decision tree analysis.
Inflammatory marker concentrations of (A) CCL11 in picograms per milliliter and (B) sCD14 in picograms per milliliter detected by fluorescent bead–based multiplex assays in endometrial cytobrushes of mares with intrauterine fluid, negative in gray (n = 38) and positive in red (n = 66) and edema, normal in gray (n = 93) and inappropriate in red (n = 22), respectively, in a prospective cross-sectional study. *Difference between groups (P < .05).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
Median and SE of the median of inflammatory marker concentrations of TNF-α (Diestrus; n = 16) and TNF-α (Estrus; n = 88) in picograms per milliliter detected by fluorescent bead–based multiplex assays in endometrial cytobrushes of mares with (A) bacterial culture, negative in gray (n = 6) and positive in red (n = 9); (B) with negative in gray (n = 14) and positive in red inflammation of cytology (n = 68); (C) Biopsy grade I or IIA in gray (n = 14) and biopsy grade IIB or III in red (n = 2); and (D) in mares negative for endometritis in gray (n = 14) and positive for endometritis in red (n = 57) in a prospective cross-sectional study. *Difference between groups (P < .05). **Difference between groups (P < .03). ***Difference between groups (P < .01).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
Median and SE of the median of inflammatory marker concentrations of CCL2, CCL5, CCL11, TNF-α, IL-1β, and sCD14 in picograms per milliliter from endometrial cytobrush samples. Estrus mares negative for endometritis are in gray (n = 57), and those positive for endometritis are in red (n = 14; positive cytology and/or positive culture).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
Decision tree for inflammatory markers in endometrial cytobrush samples
The decision tree was used in this study as a robust regression model to identify threshold concentrations of 1 or a combination of 2 or more of the 8 detectable inflammatory markers collected using the cytobrush in order to predict the probability of identifying estrous mares with endometritis as well as mares sampled in estrus that were bred to predict chances of those mares getting pregnant. The analysis demonstrated that TNF-α concentrations that were equal or above 3,942 pg/mL (found in 10 of 24 mares) identified 42% of mares with endometritis, whereas concentrations of TNF-α below 3,942 pg/mL (found in 43 of 47 mares) were associated with a 91% probability of identifying mares as not hot having endometritis. Furthermore, if concentrations of TNF-α were below 3,942 pg/mL combined with a concentration of CCL5 below 5,985 pg/mL (found in 35 of 35 mares), the chances of the mare having a healthy endometrium increased to 100% (Figure 6).
Decision tree analysis from a prospective cross-sectional study demonstrating the most predictive inflammatory marker(s) and the probability of identifying estrus mares (A) with endometritis or without endometritis and (B) likely to get pregnant when sampled in estrus just prior to being bred. Prob = Probability.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.02.0059
When pregnancy outcome (yes/no) was used in the decision tree, mares with an sCD14 concentration equal to or above 15,648 pg/mL (found in 6 of 6 mares) had a 100% chance of not getting pregnant in that cycle. When sCD14 concentration was below 1,548 pg/mL, the chances of not getting pregnant decreased to 63% (found in 24 of 38 mares).
Discussion
The ability to identify mares with endometritis reliably and accurately prior to breeding would benefit both clients and equine veterinarians alike by avoiding wasted breeding cycles of reproductively unfit mares. We identified endometrial cytobrush samples to yield higher concentrations of inflammatory markers compared to endometrial swabs overall as well as in mares with endometritis. The results from this study showed that mares with a healthy endometrium had lower TNF-α and CCL5 concentrations in endometrial cytobrush samples, making these inflammatory markers good candidates as additional screening tools for reproductive health in mares presenting for breeding. Previous research investigating diagnostic markers in endometritis showed that the combined genes of the antimicrobial peptide β-defensin 1, lysozyme, and secretory leukoprotease inhibitor had both sensitivity and specificity of 94% to diagnose mares with persistent mating-induced endometritis.22 The study by Marth et al22 measured mRNA expression from endometrial biopsies and compared susceptible-versus-resistant mares. The ability to use less-invasive diagnostic sampling methods, such as a serum or endometrial swab or cytobrush samples, on the same breeding cycle prior to breeding would be beneficial for practitioners to avoid wasted estrous cycle breeding attempts. Two other recent publications investigated markers of Arabian mares with endometritis, including serum cytokine levels, microRNA (miR), and prostaglandins in serum, and found that IL-8 and IL-1β were moderate predictors of subclinical endometritis and that miR-155, miR-223, miR-17, miR-200a, and miR-205 had higher levels of expression in old compared to young mares with endometritis.23,24 Additionally, no differences in the serum concentrations of sCD14, IL-10, or CCL2 in the serum from mares with postpartum metritis before and after uterine lavage were identified.25 A variation of results among studies can originate from the sample types used to measure the concentration of inflammatory markers either systemically or locally (serum vs endometrium), the classification of endometritis, the degree of inflammation (variations of cutoff values of neutrophils and different sampling techniques), the assays used to measure the cytokines, and the specificity of the antibodies in the assays for equine cytokines and chemokines.
Our study has shown no differences in the concentrations of inflammatory markers measured in endometrial swabs compared to endometrial cytobrush samples. Cytobrush samples overall tended to have higher marker concentrations compared to swabs, and that was also the case for mares with endometritis. In addition, there was a qualitative difference between the endometrial swab and brush samples for the inflammatory marker analysis. The marker concentrations in the swabs for sCD14 were decreased in mares with inappropriate edema, and sCD14 had decreased concentrations in mares with positive bacterial cultures. Cytobrush samples resulted in a different inflammatory marker pattern: increased CCL11 concentrations were identified in mares with uterine fluid. In addition, TNF-α concentrations were elevated during estrus in mares with positive cytology results, in mares with a higher degree of endometrial biopsy grade, and in mares with endometritis. The variations in the sampling methods can likely explain the difference in inflammatory marker results that we have observed between the 2 intrauterine sample types. While an endometrial swab collects mainly superficial fluid from the lumen of the uterus or from the upper surface of the uterine mucosa, the cytobrush can sample the endometrium slightly deeper and likely collects a less diluted mucosal secretion sample, including some endometrial epithelium and tissue immune cells. Consequently, inflammatory markers can somewhat differ depending on the sampling method. In our experience, cytobrush samples made the analysis more reliable by providing higher inflammatory marker values overall and by broadening the marker detection ranges. Hence, we would emphasize the use of cytobrush samples for further standardization of reference intervals for inflammatory markers in the uterus.
Tumor necrosis factor-α’s role in equine endometritis has been previously reviewed and shown to be increased in susceptible mares with persistent mating-induced endometritis compared to resistant mares.26 C-C motif chemokine ligand 5 is a chemokine produced by a variety of cells, including eosinophils, platelets, macrophages, and endothelial and endometrial cells, and has several roles in inflammation and angiogenesis.27 Recently, TNF-α and CCL5 were demonstrated to be increased in the uterine LVL fluid from mares with chronic endometrial fibrosis (biopsy grades IIB and III) compared to mares with healthy endometriums.16 In the current study, we identified those proinflammatory proteins to be good candidates for detecting mares with endometritis using an endometrial cytobrush.
The goal of our research was to discover potential inflammatory marker candidates with a noninvasive endometrial sampling technique to screen mares for endometritis. We were able to detect several changes in inflammatory markers in mares with acute inflammatory endometritis as well as when separately analyzing samples associated with a positive cytology or positive culture. The antibodies used for measuring the inflammatory proteins in our study are equine-specific antibodies that have been previously validated and measured with a fluorescent bead–based multiplex assay.14,15,28,29 The multiplex assay can be performed reliably and accurately, giving results within the same day, and the proteins are stable under field and transport conditions.
A limitation of this approach was the relatively small sample set that was sampled from clinical cases. These mares were often sampled in estrus as it is common among practitioners to take diagnostics in mares with an open cervix, hence the smaller number of samples in diestrus, which may affect comparing the concentrations of sCD14 and TNF-a based on the stage of the cycle. The small number of mares with a biopsy is reflective of the need for this line of research investigating minimally invasive diagnostic tests as many owners only allow for cytology and culture. Another limitation of the study is the classification of mares as susceptible or resistant to postbreeding-induced endometritis as some mares received treatment, such as oxytocin, as would be standard to treat postbreeding fluid in a clinical case. Finally, pregnancy status was determined only at the first pregnancy check (day 14 to 16 after ovulation) to provide a fertility outcome to the study as mares started to be lost to follow-up beyond the first pregnancy ultrasound. Overall, the limitations of this study reflect using clinical cases for a prospective study. On one hand, a controlled study30 of susceptible and resistant mares in a challenge model with spermatozoa would likely improve our understanding of the reproductive immunology in mares. Outside of that understanding, studies with clinical cases provide variation that would not be accounted for in a controlled study yet exists in the mares that practitioners treat daily. To further support the conclusions of this study, a larger number of mares with endometritis would be needed, including a broader variation of endometritis classifications (acute, chronic, and postmating-induced endometritis), and with histological scoring of inflammation and degeneration, such as fibrosis, as the gold standard for the final diagnosis of endometritis.
A few of the inflammatory markers, such as TNF-α and sCD14, were consistently elevated in cytobrush samples of mares with endometritis (endometrial inflammation on cytology and/or positive cultures) and in mares with negative pregnancy outcomes, respectively. The decision tree analysis has confirmed these findings and identified CCL5 and TNF-α as inflammatory marker candidates to screen mares for endometritis, even though a larger population of reproductively healthy mares should be sampled to create reference intervals. The elevation of the above inflammatory markers in mares that are deemed reproductively healthy should alert the practitioner to be suspicious and pursue further diagnostics, such as endometrial biopsy. The decision tree analysis using the pregnancy outcome should be interpreted cautiously as those were clinical cases in which a variety of treatments, such as antimicrobials, mucolytics, biofilm disruptors, uterine lavages, and others, were used after breeding to improve the odds of getting mares pregnant, potentially biasing the results.
In conclusion, inflammatory marker analysis using uterine cytobrush samples from mares that are presented for breeding is a minimally invasive technique to confirm reproductive health. Inflammatory marker analysis has a quick turnaround and can be used in addition to existing testing methods by cytology and bacterial culture to advance the differentiation of reproductively healthy mares versus those that potentially require additional diagnostics (biopsy) or treatment prior to breeding. In particular, the quantification of intrauterine CCL5 and TNF-α, and also sCD14, provides a valuable inflammatory marker set for identifying mares with a healthy endometrium and can become a valuable tool to improve breeding success rates.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to thank the clients whose mares provided samples for this study. They would also like to thank John Beeby for processing the samples through the Animal Health Diagnostic Center at Cornell University. Additionally, the authors would like to thank Stephen Parry from the Cornell Statistical Unit for the statistical analysis guidance throughout the study.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This study was carried out with the support of the Harry M. Zweig Memorial Fund for Equine Research at Cornell University and USDA/National Institute of Food and Agriculture grant No. 2019–67015-29833, “Development of Equine Immune Reagents.” This publication was supported, in part, by NIH grant No. T32GM108563 as fellowship funding for JL.
ORCID
Jennine Lection https://orcid.org/0000-0001-5173-1426
Mariana Diel de Amorim https://orcid.org/0000-0002-3385-3654
References
- 1.↑
Varadin M. Endometritis, a common cause of infertility in mares. J Reprod Fertil Suppl 1975;(23):353–356.
- 2.↑
Traub-Dargatz JL, Salman MD, Voss JL. Medical problems of adult horses, as ranked by equine practitioners. J Am Vet Med Assoc. 1991;198(10):1745–1747. doi:10.2460/javma.1991.198.010.1745
- 3.↑
Bain AM. The rôle of infection in infertility in the thoroughbred mare. Vet Rec. 1966;78(5):168–173. doi:10.1136/vr.78.5.168
- 4.↑
Morris LHA, Allen WRR. Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket. Equine Vet J. 2002;34(1):51–60. doi:10.2746/042516402776181222
- 5.↑
Rasmussen CD, Petersen MR, Bojesen AM, Pedersen HG, Lehn-Jensen H, Christoffersen M. Equine infectious endometritis—clinical and subclinical cases. J Equine Vet Sci. 2015;35(2):95–104. doi:10.1016/j.jevs.2014.12.002
- 6.↑
Cocchia N, Paciello O, Auletta L, et al. Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares. Theriogenology. 2012;77(1):89–98. doi:10.1016/j.theriogenology.2011.07.020
- 7.↑
Overbeck W, Witte TS, Heuwieser W. Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenology. 2011;75(7):1311–1318. doi:10.1016/j.theriogenology.2010.12.002
- 8.↑
de Amorim MD, Gartley CJ, Foster RA, et al. Comparison of clinical signs, endometrial culture, endometrial cytology, uterine low-volume lavage, and uterine biopsy and combinations in the diagnosis of equine endometritis. J Equine Vet Sci. 2016;44:54–61. doi:10.1016/j.jevs.2015.10.012
- 9.↑
Holyoak GR, Premathilake HU, Lyman CC, et al. The healthy equine uterus harbors a distinct core microbiome plus a rich and diverse microbiome that varies with geographical location. Sci Rep. 2022;12(1):14790. doi:10.1038/s41598-022-18971-6
- 10.↑
LeBlanc MM, Magsig J, Stromberg AJ. Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares. Theriogenology. 2007;68(3):403–412. doi:10.1016/j.theriogenology.2007.04.038
- 11.↑
Card C. Post-breeding inflammation and endometrial cytology in mares. Theriogenology. 2005;64(3):580–588. doi:10.1016/j.theriogenology.2005.05.041
- 12.↑
Kenney RMR, Doig PA. Equine endometrial biopsy. In: Morrow D, ed. Current Therapy in Theriogenology. 2nd ed. Saunders; 1986:723–729.
- 13.↑
Perkins G, Babasyan S, Stout AE, et al. Intranasal IgG4/7 antibody responses protect horses against equid herpesvirus-1 (EHV-1) infection including nasal virus shedding and cell-associated viremia. Virology. 2019;531:219–232. doi:10.1016/j.virol.2019.03.014
- 14.↑
Wagner B, Freer H. Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses. Vet Immunol Immunopathol. 2009;127(3–4):242–248. doi:10.1016/j.vetimm.2008.10.313
- 15.↑
Wagner B, Ainsworth DM, Freer H. Analysis of soluble CD14 and its use as a biomarker in neonatal foals with septicemia and horses with recurrent airway obstruction. Vet Immunol Immunopathol. 2013;155(1–2):124–128. doi:10.1016/j.vetimm.2013.05.018
- 16.↑
Lection JM, Wagner B, Miller AD, et al. Inflammatory protein biomarkers in the low-volume lavage of mares with endometritis and degenerative endometrial fibrosis. In: Proceedings of the 66th Annual Convention of the American Association of Equine Practitioners. American Association of Equine Practitioners; 2020:150.
- 17.↑
Riddle WT, LeBlanc MM, Stromberg AJ. Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice. Theriogenology. 2007;68(3):395–402. doi:10.1016/j.theriogenology.2007.05.050
- 18.↑
Lection J, Wagner B, Byron M, et al. Inflammatory markers for differentiation of endometritis in the mare. Equine Vet J. 2024;56(4):678-687. doi:10.1111/evj.14058
- 19.↑
Berk RA. Statistical Learning from a Regression Perspective. 3rd ed. Allen G, De Veaux R, Nugent R, eds. Springer Nature Switzerland; 2016.
- 20.↑
Rokach L, Maimon O. Classification trees. In: Maimon O, Rokach L, eds. Data Mining and Knowledge Discovery Handbook. 2nd Ediition. Springer US; 2005:149–174.
- 21.↑
James G, Witten D, Hastie T, Tibshirani R. Tree-based methods. In: An Introduction to Statistical Learning with Applications in R. Springer US; 2021:327–365.
- 22.↑
Marth CD, Firestone SM, Hanlon D, et al. Innate immune genes in persistent mating-induced endometritis in horses. Reprod Fertil Dev. 2018;30(3):533–545. doi:10.1071/RD17157
- 23.↑
Hedia M, Ibrahim S, Mahmoud K, Ahmed Y, Ismail S, El-Belely M. Hemodynamic changes in cytokines, chemokines, acute phase proteins and prostaglandins in mares with subclinical endometritis. Theriogenology. 2021;171:38–43. doi:10.1016/j.theriogenology.2021.05.011
- 24.↑
Ibrahim S, Hedia M, Taqi MO, et al. Alterations in the expression profile of serum miR-155, miR-223, miR-17, miR-200a, miR-205, as well as levels of interleukin 6, and prostaglandins during endometritis in Arabian mares. Vet Sci. 2021;8(6):98. doi:10.3390/vetsci8060098
- 25.↑
Tukia E, Wagner B, Vainio K, Mönki J, Kareskoski M. The effect of uterine lavage on soluble CD14, chemokine ligand 2, and interleukin 10 levels in mares with postpartum metritis. J Equine Vet Sci. 2021;98:103365. doi:10.1016/j.jevs.2020.103365
- 26.↑
Canisso IF, Segabinazzi LGTM, Fedorka CE. Persistent breeding-induced endometritis in mares - a multifaceted challenge: from clinical aspects to immunopathogenesis and pathobiology. Int J Mol Sci. 2020;21(4):1432. doi:10.3390/ijms21041432
- 27.↑
Marques RE, Guabiraba R, Russo RC, Teixeira MM. Targeting CCL5 in inflammation. Expert Opin Ther Targets. 2013;17(12):1439–1460. doi:10.1517/14728222.2013.837886
- 28.↑
Schnabel CL, Wemette M, Babasyan S, et al. C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies. Vet Immunol Immunopathol. 2018;204:28–39. doi:10.1016/j.vetimm.2018.09.003
- 29.↑
Schnabel CL, Babasyan S, Freer H, Larson EM, Wagner B. New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells. Vet Immunol Immunopathol. 2021;238:110284. doi:10.1016/j.vetimm.2021.110284
- 30.↑
Troedsson MH. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology. 1999;52(3):461–471. doi:10.1016/S0093-691X(99)00143-0
