Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

In the past, most research in equine reproduction has been performed in vivo but the use of in vitro and ex vivo models has recently increased. This study aimed to evaluate the functional stability of an ex vivo hemoperfused model for equine uteri with molecular characterization of marker genes and their proteins. In addition, the study validated the respective protein expression and the aptness of the software QuPath for identifying and scoring immunohistochemically stained equine endometrium. After collection, uteri (n = 12) were flushed with preservation solution, transported to the laboratory on ice, and perfused with autologous blood for 6 h. Cycle stage was determined by examination of the ovaries for presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Samples were obtained directly after slaughter, after transportation, and during perfusion (240, 300, 360 min). mRNA expression levels of progesterone (PGR), estrogen (ESR1) and oxytocin (OXTR) receptor as well as of MKI67 (marker of cell growth) and CASP3 (marker of apoptosis) were analyzed by RT-qPCR, and correlation to protein abundance was validated by immunohistochemical staining. Endometrial samples were analyzed by visual and computer-assisted evaluation of stained antigens via QuPath. For PGR, effects of the perfusion and cycle stage on expression were found (P < 0.05), while ESR1 was affected only by cycle stage (P < 0.05) and OXTR was unaffected by perfusion and cycle stage. MKI67 was lower after 360 min of perfusion as compared to samples collected before perfusion (P < 0.05). For CASP3, differences in gene expression were found after transport and samples taken after 240 min (P < 0.05). Immunohistochemical staining revealed effects of perfusion on stromal and glandular cells for steroid hormone receptors, but not for Ki-67 and active Caspase 3. OXTR was visualized in all layers of the endometrium and was unaffected by perfusion. Comparison of QuPath and visual analysis resulted in similar results. For most cell types and stained antigens, the correlation coefficient was r > 0.5. In conclusion, the isolated hemoperfused model of the equine uterus was successfully validated at the molecular level, demonstrating stability of key marker gene expression. The utility of computer-assisted immunohistochemical analysis of equine endometrial samples was also confirmed.

Evaluation of an ex vivo model of the blood-perfused equine uterus.

Uterine pathologies are the most common causes of infertility in mares. This study aimed to establish an ex vivo blood-perfused model for equine uteri and investigate the possible effects of different cycle stages (estrus, diestrus and anestrus) on the applicability of the model. Uteri (n = 13) were collected at an abattoir, flushed with preservation solution, transported to the laboratory on ice, and isolated perfused with autologous blood for 6 h (n = 12). For negative control, one uterus was handled as described but left without perfusion for 6 h. The cycle stage was determined by examination of the ovaries for the presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Sonomicrometry crystals were implanted into the myometrium to record spontaneous contractions and the response to 0.5 IU oxytocin after 6 h of perfusion. Analyses of the arterial and venous perfusate were performed every hour to determine glucose consumption, lactate production, pH, lactate dehydrogenase activity (LDH), and potassium concentration (K+). Biopsy samples were obtained directly after slaughter, after transportation, after equilibration, after 4, 5, and 6 h of perfusion, and immediately after removal from the perfusion system. The uteri's glucose consumption and lactate production increased over time (p < 0.05), but no differences among cycle stages were detected. pH (arterial and venous) increased over time (p < 0.05). No changes for LDH were observed. K+ increased after 4 h of perfusion (p < 0.05), but was unaffected by the cycle stage. Spontaneous contractions were present in all perfused uteri, but myometrial activity in the negative control was limited to the 2nd hour of perfusion. No effects of cycle stage on contraction amplitude and duration after oxytocin administration were detected. The cycle stage did not affect frequency (except after 5 h of perfusion), amplitude, duration of contractions, or edema formation. Histology revealed congestion of endometrial capillaries after 4, 5, and 6 h perfusion time. In conclusion, the ex vivo model was capable of supporting the functionality of equine uteri for 6 h. However, viability and histomorphology of the endometrium appeared to be impaired after 4 h of perfusion. Effects of the cycle stage on the applicability of the model were absent.

Occurrence of Intrauterine Purulent Concrements in a Maiden Mare-A Case Report.

Pyometra is an uncommon condition in mares associated with various symptoms. Here, we report a case of a 13-year-old Icelandic barren maiden mare with recurrent vaginal discharge. Ultrasonographically, the mare displayed intrauterine spherical masses of inhomogenous texture, which were identified as purulent concrements in hysteroscopy. The purulent concrements were successfully removed via uterine lavage after endoscope-assisted comminution. Microbiologic examination of the concrements revealed growth of Streptococcus equi subspecies zooepidemicus, Actinobacillus species, Pseudomonas aeruginosa, Staphylococcus intermedius, Pseudomonas fulva, Citrobacter freundii, and Chryseobacterium species. Systemic antibiotic treatment with trimethoprim-sulfadiazine and additional uterine lavages were performed for 10 days. A follow-up examination revealed absence of intrauterine masses but reoccurrence of pyometra due to an impatent cervical canal. The pyometra condition was resolved by insertion of a cervical stent for prevention of intrauterine fluid accumulation. In conclusion, uterine masses, which may severely impact fertility, are best diagnosed by hysteroscopy. Intrauterine purulent concrements should be considered as an atypical form of equine pyometra.