Renal biomarkers, clinical parameters, and renal Doppler velocimetry in bitches with cystic endometrial hyperplasia-pyometra complex.

Cystic endometrial hyperplasia (CEH)-pyometra complex is the most common uterine infection in adult and elderly bitches and can cause renal dysfunction. The aim of this study was to measure and compare urinary creatinine, urea, symmetric dimethylarginine (SDMA), urinary protein-creatinine ratio (UPC), measurement of systolic blood pressure (SBP), and Doppler velocimetry of renal arteries in patients with CEH-pyometra complex before and after an average of 6 months of treatment, evaluating the possibility of the changes persisting. The evaluation was conducted at two moments: M1 (at the diagnosis of CEH-pyometra, n = 36) and M2 (after an average of six months of treatment, n = 16). For the control group, eight bitches with no changes in blood tests or history of conditions underwent Doppler ultrasound evaluation of the renal arteries. At both M1 and M2, we measured creatinine, urea, and serum SDMA, UPC, SBP, and Doppler ultrasound of the renal arteries. Patients were evaluated according to the following groups: azotemic (AZO) and non-azotemic (NAZO), and open and closed cervix pyometra. The parameters were compared between animals present in both moments presented as M1R (bitches that were in M1 and M2) and M2. Statistical significance was considered when p < .05. The medians found for creatinine in M1 were as follows: 1.15 mg/dL, being 1.8 mg/dL for AZO (12/36) and 0.95 mg/dL for NAZO (24/36); and in M2: 0.85 mg/dL (16/16), being 1.15 mg/dL for AZO (4/16) and 0.8 mg/dL for NAZO (12/36). For urea, in M1 it was 36 mg/dL (32/36), with AZO being 103 mg/dL (11/32) and 33 mg/dL in NAZO (21/32); and in M2 32 mg/dL (16/ 16), being 29 mg/dL for AZO (4/36), and 31 mg/dL for NAZO (3/15). The median SDMA measured in M1R was 17 µg/dL (15/16), with AZO being 31 µg/dL (3/15), and NAZO being 16.5 µg/dL (12/15); and in M2, SDMA was 12 µg/dL (16/16), with AZO being 12.5 µg/dL (4/16), and NAZO being 12 µg/dL (12/16). The median UPC measured in M1 was 1.15 (10/36), with AZO being 0.25 (1/10), and NAZO being 1.38 (9/10); and in M2, it was 0.2 (13/16), being 0.1 in AZO (4/13), and 0.2 (9/16) in NAZO. For SBP, in M1, it was 118 mmHg (30/36), with AZO being 102 mmHg (10/30) and 133 mmHg in NAZO (20/30); and in M2 142.5 mmHg (12/16), being 155 mmHg for AZO (4/12), and 140 mg/dL for NAZO (8/12). When comparing animals with open and closed cervixes, a difference was found between SDMA measurements (p = .001). There was a distinction between PI and RI of the left and right kidneys consecutively (p = .007; p = .033; p = .019; p = .041). Correlations found in M1: SDMA × PI RIM DIR (r = 0.873; p = .002), SDMA × PSV RIM ESQ (r = 0.840; p = .004), SDMA × EDV RIM ESQ (r = 0.675; p = .046). With this study, we conclude a return to normality of renal biomarkers and clinical parameters after six months. Yet, there is a persistence of Doppler velocimetric measurements between the two moments. Thus, this parameter is not suitable for identifying and classifying chronic kidney injury in bitches with pyometra.

Causes of pregnancy arrest in the canine species.

In the canine species, early pregnancy arrest before 30-40 days of pregnancy will induce intra-uterine embryonic or foetal resorption, with very few clinical signs. If no genital examination by ultrasound is performed at that time, it will often remain unnoticed and the bitch will be qualified as infertile. It is only when pregnancy stops at a later stage, mostly after 40 days, that clinical signs will be seen. Expulsion of aborted foetuses or placentas may be observed, although the dam frequently eats the expelled tissues. Intra-uterine mummification may also occur. This article reviews the causes of pregnancy termination in the bitch, both at the embryonic and the foetal stages, reported in the literature. Canine brucellosis is by far the most important disease concerned in this aspect. There is an important current concern about this disease, due to the fact that several outbreaks have been observed recently in Europe, and that it is a very contagious disease; it may be an underestimated zoonosis. Other bacterial causes of pregnancy arrest are sporadic. There is, however, a growing attention towards the microbiological content of raw food diets which are more and more popular among dog breeders and may-if badly prepared-contain abortifacient bacteria such as Campylobacter jejuni or Listeria monocytogenes. The abortifacient role of endogenous vaginal bacteria and mycoplasms remains unclear and may be related to an imbalanced vaginal flora with subsequent ascending bacterial contamination of the uterus. The role of Canine Herpesvirus on abortion is controversial and probably not frequent. Other viruses have been shown experimentally to induce abortion but their natural occurrence in this respect remains unknown. The same applies to the parasite Neospora caninum which is suspected, but not proven, potentially to induce pregnancy arrest in bitches. Among non-infectious causes, uterine pathology such as cystic endometrial hyperplasia (CEH) or sub-clinical post-mating endometritis which can cause infertility and may also induce embryonic resorption. The role of luteal insufficiency in pregnancy arrest is probably overestimated.

Endometrial hyperplasia and pyometra in captive lions (Panthera leo) and tigers (Panthera tigris).

Endometrial hyperplasia (EH) is a pathologic condition of the uterus with increased endometrial gland to stroma ratio compared to normal cyclic uterine proliferation. In domestic animals, EH often involves cystic distension of proliferating endometrial glands and may be concurrent with pyometra. In large captive nondomestic felids, an association between EH and pyometra is common; however, detailed species differences between the histological uterine findings in lions (Panthera leo) and tigers (Panthera tigris) and clinical manifestations have yet to be described. Uterine sections from 14 lions and 24 tigers with EH and/or pyometra were scored for several histological parameters and clinical histories were recorded. The percentage of endometrium affected by hyperplasia, endometrial gland to stroma ratio, and adenomyosis were significantly (P = .0385, P = .0008, and P = .0463, respectively) more severe in lions compared to tigers as univariate analytes. Although tubular complexity was not statistically significant (P = .3254), when combined as a proposed EH grading scheme, these 4 features confirmed lions had significantly (P = .0068) more severe EH compared to tigers. Endometrial hyperplasia severity significantly correlated with inflammation/pyometra severity when controlling for species (P = .0203). A significant correlation exists between pyometra-associated clinical sign severity and the presence of pyometra in tigers, (P = .0026) but not in lions (P = .1144). There was no statistical difference in the severity of clinical signs associated with pyometra between these species (P = .1986). This proposed grading scheme may have clinical utility in providing a more consistent and objective evaluation of EH in large captive felids.

Peroxisome proliferator-activated receptor expression in the canine endometrium with cystic endometrial hyperplasia-pyometra complex.

The most common uterine diseases in bitches occurring during diestrus are cystic endometrial hyperplasia (CEH) and pyometra. These diseases can coexist as CEH-pyometra complex (CEH-P). Their pathogenesis has not been fully explained. Peroxisome proliferator-activated receptors (PPARs) are important factors regulating mammalian reproductive function and inflammatory processes. Although there is a lack of data concerning the expression of PPARs in the canine endometrium during CEH and CEH-P, we hypothesized that they might be involved in the development of pathological disorders of the canine endometrium. Therefore, the current study was conducted to evaluate and compare PPARα, PPARδ and PPARγ mRNA expression using quantitative real-time PCR (qPCR) and their immunolocalization using immunohistochemistry (IHC) staining in the endometrium of clinically healthy bitches (control group; n = 8) and those with CEH (n = 8) or CEH-P (n = 8). For quantification, the arithmetic means of all intensities of immunostaining from the cells were measured with the optical density. PPARα, PPARδ and PPARγ were detected in the luminal epithelium, glandular epithelium and stromal cells. The mRNA transcription of PPARα was higher in the CEH group than in the control group (p < .05). Additionally, the mRNA expression and immunostaining intensities of PPARδ and PPARγ in the endometrium in the CEH-P group were downregulated relative to those in the control group (p < .05). Moreover, the serum progesterone concentration measured by direct radioimmunoassay was decreased in the CEH-P group compared to the control group (p < .001) and CEH group (p < .05). The obtained results indicate that PPARs are present in the canine endometrium and that their mRNA profile and intensity levels change under pathological conditions such as CEH and CEH-P. This finding may suggest a correlation between changes in the PPAR expression profile and hormonal disturbances, as well as the potential involvement of PPARs in signal transduction during inflammatory processes occurring in the endometrium during CEH-P. These results pave the way to further research into the role of PPARs in the pathogenesis of CEH and CEH-P in female dogs.

Doppler ultrasonographic evaluation of medically treated female dogs with cystic endometrial hyperplasia-pyometra complex.

Monitoring the treatment outcome of canine cystic endometrial hyperplasia-pyometra complex (CEH-P) is essential to identify nonresponding patients who might require a rapid intervention to avoid life-threatening conditions. Uterine artery Doppler characterization may contribute to monitoring medically treated CEH-P, but published studies are currently lacking. Therefore, the aim of this longitudinal prospective observational study was to evaluate uterine artery resistance changes in the medical treatment of female dogs with CEH-P. Twelve diestrous female dogs suffering from CEH-P were treated with a combined protocol of cabergoline, cloprostenol, and antibiotics. The animals were clinically and ultrasonographically evaluated before the beginning of treatment (day 0) and on days 3, 7, 14, 21, and 28. The widest transverse diameter and luminal diameter of uterine horns were measured, as well as the peak systolic velocity (PSV) and end diastolic velocity (EDV) of uterine arteries. The resistance index (RI), pulsatility index (PI), and notch deep index (NDI) were calculated. On day 3, nine of 12 animals showed clinical improvement. In all these female dogs, the widest transverse sectional diameter (P < 0.01), luminal diameter (P < 0.01), PSV (P < 0.01), and EDV (P < 0.01) progressively decreased, while RI (P < 0.01), PI (P < 0.01), and NDI (P < 0.01) increased up to day 21. In nonresponding dogs (3/12), Doppler parameters remained unchanged. Although comparisons of PSV, EDV, and NDI were significantly different, these results should be cautiously interpreted due to the low statistical power. female dogs that responded to this treatment showed an increase in uterine artery resistance, along with clinical and ultrasonographic improvement.

Morphological changes in bitches endometrium affected by cystic endometrial hyperplasia - pyometra complex - the value of histopathological examination.

BACKGROUND: Cystic endometrial hyperplasia-pyometra complex (CEH-P) is one of the most common uteropathies in bitches. In diseases with mild or obscure clinical signs and normal uterine size, a diagnosis based on a clinical assessment might be incorrect. The main aim of the research was to determine the morphological variables accompanying uterine diseases in bitches in microscopic evaluation. Consequently, the obtained results can be used to create a new classification system for uterine pathological changes during the development of the CEH-P, diagnosed by microscopic examination in bitches. Material for the study consisted of the uteri of 120 female dogs, aged 1-16 years, obtained during routine ovariohysterectomies. Macroscopic observation after a longitudinal incision of the uterine horns, allowed a preliminary classification of the uteri into research groups: control group (physiological uteri), and groups GI-III uteri collected form bitches with varying degrees of endometrial pathology. These preliminary classifications were then verified by histological analysis (H&E stain). RESULTS: The obtained results made it possible to determine and describe the prevalence (%) of pathological changes characteristic of the analyzed uterine diseases in the examined bitches. Histopathological analyses that were conducted have confirmed preliminary macroscopic evaluation for the control group, group GII (CEH), and group GIII (pyometra). In the uteri of the GI group, a severe congestion of the endometrium has been observed - this is typical of inflammation - which was not confirmed during histopathological examinations. However, these examinations revealed acute endometrial haemorrhage of varying severity. CONCLUSIONS: Early reproduction disorders in bitches are, in general, not confirmed by clinical signs in the examined animals. The results show that during classification of typical morphological changes in the endometrium over the development of the CEH-P complex in bitches microscopic examinations are required. The obtained results indicate a frequent lack of consistency in the macroscopic assessment and histological analysis of the endometrium, observed in the analyzed uterine diseases, which in most cases is not followed by clinical symptoms. The presented classification of uterine diseases may be useful as a diagnostic tool in reproductive disorders in bitches and in examination in the field of basic research.

Investigation of the usability of kisspeptin and oxidative stress parameters in the early diagnosis of asymptomatic cystic endometrial hyperplasia in dogs.

This study aimed to investigate the differences in oxidative stress index (OSI) and kisspeptin levels in clinically asymptomatic dogs with cystic endometrial hyperplasia (CEH) compared to healthy and pregnant dogs, and to determine the usability of the obtained results in the diagnosis of asymptomatic CEH. The study comprised three groups; a healthy (n = 8), a pregnant (n = 10) and a CEH (n = 10). All dogs in the three groups were included in the study at the 30 ± 3th day after estrus, and blood samples were collected for analysis of kisspeptin, total antioxidant status (TAS), total oxidant status (TOS), progesterone (P4), estradiol (E2) and some biochemical parameters (TSH; thyroid stimulating hormone, ALT; alanine aminotransferase, AST; aspartate aminotransferase, ALP; alkaline phosphatase, LDH; lactate dehydrogenase, CRE; creatine and BUN; blood urea nitrogen). In addition, OSI value was calculated. P4 and ALT and BUN levels were significantly lower and higher in CEH group than the pregnant group, respectively (p < .05). While kisspeptin and TAS levels were significantly lower in CEH group compared to the healthy and pregnant groups (p < .01), OSI level increased dramatically. In conclusion, it was confirmed that CEH clearly affected kisspeptin and OSI levels, and it is thought that these parameters may be an alternative diagnostic tool for the detection of CEH after further studies.

A descriptive angiographic study of the uterine arteries during pregnancy, the postpartum period and CEH/pyometra in bitches.

The aim of this descriptive study was to monitor the changes in uterine arteries during pregnancy, postpartum period and pyometra in bitches using angiography. Fifteen uteri of mixed breed bitches on days 24, 30, 33, 40, 43, 47, 50 and 56 of pregnancy and weeks 1, 2, 3, 4 and 7-8 of postpartum and two CEH/pyometra bitches were examined after ovariohysterectomy. The results showed that with the onset of normal pregnancy and in about 30 ± 1 days of gestation, anastomoses begin to form between the left and right middle uterine arteries, developing during the next days and continuing until 4 weeks postpartum. On 4th week after parturition, when physiologic changes occur and the uterus returns to non-pregnant conditions, these anastomoses begin to degenerate, and they completely disappear approximately on the 7th-8th week after parturition. Similarly, in CEH/pyometra bitches, anastomoses were formed between left and right median uterine arteries. These findings can be considered as a part of the physiological changes of the uterus and its vessels during pregnancy and postpartum periods and could affect the results and interpretation of relevant findings.

Ultrasonographic appearance of pseudo-placentational endometrial hyperplasia in a dog.

A 5-year-old, clinically normal intact female Miniature Schnauzer was presented for demonstrative ultrasonography in a seminar. She had two pregnancies in the past and had a natural mating 2 months previously. Ultrasonography revealed a segmental and circumferential mural thickening of the right uterine horn. The endometrium was markedly thickened with multiple organized hyperechoic linear striations, perpendicular to the mucosal surface. Histology revealed focal endometrial hyperplasia resembling the maternal tissue of the normal canine placenta. A diagnosis of spontaneous pseudo-placentational endometrial hyperplasia (PEH) was made. This study described a unique ultrasonographic appearance of PEH, which may facilitate the diagnosis of PEH.

Plasma concentration and uterine and ovarian expressions of insulin-like growth factor-2 in dogs with cystic endometrial hyperplasia-pyometra.

The objective of this study was to investigate the plasma concentrations of insulin-like growth factor-2 (IGF-2) as well as its expression in the uterus and ovary of healthy dogs and those with cystic endometrial hyperplasia (CEH)-pyometra complex. Group 1 (n = 10) included bitches with open cervix pyometra, while Group 2 (n = 7) consisted of clinically healthy bitches in dioestrus. The number of IGF-2 immunopositive interstitial cells was significantly higher in Group 1, whereas in Group 2 there were only two cases in which a few cells were IGF-2 immunopositive. IGF-2 immunopositivity was observed in the endometrial glandular epithelium in both groups. Additionally, interstitial fibroblasts and macrophages in the endometrium were also positive in Group 1. The concentration of plasma IGF-2 was higher in Group 1 than in Group 2 (P < 0.05). The concentration was positively correlated with IGF-2 expression in the endometrial glands (r = 0.926; P < 0.001) in Group 1. However, a negative correlation was present between plasma IGF-2 concentration and IGF-2 expression in the interstitial endocrine cells of the ovary in Group 1 (r = -0.652; P < 0.05). The results suggest that IGF-2 plays an important role during the inflammatory process occurring in bitches with CEH-pyometra complex as well as in the endometrium of healthy bitches in dioestrus.

Assessment of the optimal age for a preventive ultrasonographic screening of the uterine health in bitches.

Studies about prevalence of uterine pathologies in bitches are scarce. Although correlation between age and uterine disorders was documented, the most suitable age for a preventive sonographic screening has not been proposed yet. Present study aimed to estimate the eligible age for an ultrasonographic screening of uterine abnormalities in dogs. Data regarding ultrasound examination and clinical records of non-pregnant intact females were retrospectively analysed. The age of each bitch was expressed as age ratio (actual/maximum age expected for the respective breed). The cut-off age ratio was determined by a ROC curve for overall uterine abnormalities. Frequencies of different abnormalities below and over the cut-off derived from the ROC curve were calculated and statistically analysed by chi-Square and OR. Prevalence of three categories of ultrasonographic findings was as follows: cystic endometrial hyperplasia (CEH) 18%; uterine collections (UC) 10.5%; masses (M) 1.3%. By the cut-off age ratio (0.325), derived by ROC curve (AUC = 0.91; SP 84.23%; SE 79.2%; PPV 83.4%; NPV 80%), 228 cases were divided into two subgroups: bitches over (exposed group: n.83) and below cut-off (control group: n.145). All abnormalities resulted more frequent in exposed group: OR was 24.96 (p < 0.0001: 71.1% over vs. 9% below cut-off) for overall abnormalities; 13.68 (p < 0.0001: 40.9% vs. 4.8%) for CEH; 6.13 (p < 0.002: 21.7% vs. 4.1%) for UC; 12.65 (p = 0.09: 3.6% vs. 0%) for M. Cystic endometrial hyperplasia represents the most common finding in adult bitches, followed by UC. A preventive sonographic screening for uterine abnormalities should start from 33% of expected longevity to preventively select animals requiring further evaluations.

IMAGING DIAGNOSIS-ENDOMETRIAL MINERALIZATION IN A DOG.

A 9-year-old intact female mixed breed dog was presented for mammary gland tumor surgery, and preoperative radiographs showed a tubular soft tissue opacity mass with multifocal mineralization in the caudoventral abdominal cavity. Subsequent ultrasonography demonstrated uterine dilation with echogenic fluid and endometrial acoustic shadowing consistent with mineralization. Resection of mammary gland tumors and ovariohysterectomy were performed. Pyometra was diagnosed on cytologic examination of uterine fluid. Histopathology of the uterine tissue confirmed a diagnosis of cystic endometrial hyperplasia with uterine mineralization.

ANTEMORTEM DIAGNOSIS OF CYSTIC ENDOMETRIAL HYPERPLASIA AND SUCCESSFUL OVARIOHYSTERECTOMY IN AN AFRICAN WARTHOG (PHACOCHOERUS AFRICANUS).

During a routine geriatric examination on a 9-yr-old, nulliparous female African warthog (Phacochoerus africanus), a severely enlarged, mixed echogenicity uterus was discovered during transabdominal ultrasound. Ovariohysterectomy (OVH) was elected and performed due to concern for a potential neoplastic condition. Postoperatively, the warthog was treated prophylactically with metoclopramide and ranitidine to promote gastrointestinal motility and prevent postoperative ileus. The gross appearance and histopathology confirmed the presence of extensive widely disseminated cystic changes consistent with cystic endometrial hyperplasia (CEH) and multiple para-ovarian cysts. Uterine pathology, including CEH, is a common condition in domestic potbellied pigs (Sus scrofa), and as a result, it is recommended to perform an OVH at an early age. The advanced uterine changes may have accounted for this animal's history of infertility. This case provides evidence that nulliparity may be associated with the development of uterine pathology in warthogs. Increased knowledge of reproductive pathology will aid in improved management strategies for breeding nondomestic suids.

Measurement of C-reactive protein and prostaglandin F2α metabolite concentrations in differentiation of canine pyometra and cystic endometrial hyperplasia/mucometra.

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C-reactive protein (CRP) and prostaglandin F2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.

Factors associated with uterine endometrial hyperplasia and pyometra in wild canids: implications for fertility.

The ability to safely and effectively manage reproduction is central to the success of AZA captive-breeding programs. Although the AZA Wildlife Contraception Center routinely monitors contraceptive safety, there have been no studies that compare the effects of contraceptive use to separation of males from females, the other option for preventing reproduction. We used retrospective medical records and pathology reports submitted by AZA and related facilities for the seven AZA-managed canid species to assess rates of uterine pathology relative to female reproductive life histories. Our results showed that the prevalence of both pyometra and endometrial hyperplasia (EH) was associated not only with treatment with the two most common contraceptives (Suprelorin® and MGA implants) but also with the number of years barren (i.e., not producing a litter and not contracepted). Rates of pyometra and EH were especially high in African painted dogs and red wolves, but lowest in swift and fennec foxes. The number of years producing a litter had a low association, suggesting it could be protective against uterine pathology. A more recently developed Suprelorin® protocol using Ovaban® to prevent the initial stimulation phase, followed by implant removal when reversal is desired, may be a safer contraceptive option. These results concerning the relationship between reproductive management and uterine health have important implications for AZA-managed programs, since the unsustainability of many captive populations may be due at least in part to infertility. Managing a female's reproductive lifespan to optimize or maintain fertility will require a reconsideration of how breeding recommendations are formulated.

Evaluation of uterine antioxidants in bitches suffering from cystic endometrial hyperplasia-pyometra complex.

Cystic endometrial hyperplasia-pyometra complex (CEH-P) is a common disease in sexually mature bitches. Disease progression leads to oxidative stress, resulting in the depletion of uterine antioxidants and lipid peroxidation of associated cells, which further aggravates the condition. The concentration of antioxidant enzymes, the level of lipid peroxidation within the uterine tissue, and its reflection in the serum and urine need to be elucidated. The aim of this study was to analyze the concentration of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and the lipid peroxidation marker malonaldehyde (MDA) in three types of samples, i.e., serum, urine, and uterine tissue. For this purpose, 58 pyometra-affected and 44 healthy bitches were included in the present study. All animals underwent ovariohysterectomy (OVH). Our data indicated highly significant difference (p<0.01) in the antioxidant concentrations of uterine, serum and urine samples. Furthermore, there was a highly significant (p<0.01) difference in the serum levels of ferric reducing antioxidant power (FRAP) and free radical scavenging activity (FRSA) indicated poor capacity to overcome oxidative stress in the CEH-Pyometra condition. We showed that CEH-P induces oxidative stress, which further depletes the antioxidant enzyme reserves in the uterus. Thus, the weak antioxidant defence predisposes to uterine damage and disease progression. The simultaneous depletion of antioxidants and an increase in lipid peroxidation in the serum and urine may also act as early indicators of uterine pathology.

Expression and localization of the neuropeptide phoenixin-14 and its receptor GRP173 in the canine reproductive organs and periovarian adipose tissue.

Phoenixin-14 (PNX-14) is a regulatory neuropeptide encoded by the SMIM20 gene, which has been implicated in the reproductive cycle by modulating the hypothalamic-pituitary-gonadal (HPG) axis. Recently, we showed that PNX-14 is downregulated in bitches with cystic endometrial hyperplasia and pyometra. The objective of this study was to determine the expression of Smim20, PNX-14, and its putative receptor GRP173 in the canine ovary (both healthy and those with ovarian cysts), periovarian adipose tissue (PAT) and in the endometrium during the oestrous cycle. The expression was analysed by RT-qPCR and Western blot. In tissue sections, peptides were localised by immunofluorescent assays, and blood plasma concentrations of PNX-14 were detected by EIA. The results demonstrated increased levels of PNX in bitches in the anestrus groups compared to diestrus animals. The expression of GPR173 increased in PAT during the diestrus phase and endometrial tissue in late diestrus bitches. In the ovary, strong signals of PNX-14 and GPR173 were detected in the luteal and follicular cells. Furthermore, bitches with cystic ovaries were characterised by elevated circulating PNX levels and a significantly higher expression of PNX and GPR173 in gonadal tissues, when compared with healthy animals. Moreover, a positive correlation between PNX and progesterone in the blood of healthy bitches was noted, which changed to a negative correlation in females affected by cystic ovaries. These studies expand the knowledge regarding the expression and localization of the PNX/GRP173 system in canine reproductive organs during physiological and pathological conditions.

Cystic endometrial hyperplasia-pyometra syndrome impairs the preantral follicle reserve in domestic bitches (Canis familiaris).

Cystic endometrial hyperplasia (CEH)-pyometra syndrome is the most common uterine pathological condition reported in breeding bitches, however, their described effects on fertility are limited to uterine disorders and conception rates. As the preantral follicle population represents the available reserve of gametes recruited during the lifespan, the aim of this study was to evaluate the effects of CEH-pyometra syndrome on the: (i) preantral follicle morphology, (ii) developing follicle rates, and (iii) preantral follicle and stromal cell densities. Ovarian fragments from bitches subjected to elective or therapeutic ovariohysterectomy were allocated according to uterine diagnosis as follows: control (n = 7, clinically healthy), CEH-mucometra (n = 8, uterine lumen filled with a sterile mucus), and pyometra (n = 17, presence of a purulent mucus) groups. Overall, the control group had 3.4 and 4.1-fold higher probability (P < 0.0001) of the presence of normal preantral follicles compared with CEH-mucometra and pyometra groups, respectively. Moreover, ovarian fragments from the pyometra group showed an increase in the percentage of developing follicles (P < 0.05) compared to the control. Both CEH-mucometra and pyometra groups showed lower (P < 0.05) preantral follicle and stromal cell densities (P < 0.05) compared to the control. In summary, the CEH-pyometra syndrome decreased the percentage of morphologically normal follicles and enhanced the developing follicle rates. Additionally, a reduction of preantral follicle and stromal cell densities suggests that the inappropriate uterine environment induced by CEH-pyometra syndrome can lead to premature depletion of ovarian reserve.

Uterine issues in infertile queens: Nine cases.

This prospective case series investigated potential uterine causes of infertility in queens. Purebred queens with infertility (failure to conceive, embryonic death, or failure to maintain pregnancy and produce viable kittens), but no other reproductive disorders were examined approximately 1-8 weeks before mating (Visit 1), 21 days after mating (Visit 2), and 45 days after mating (Visit 3) if pregnant at Visit 2. Investigations included vaginal cytology and bacteriology, urine bacteriology, and ultrasonography. At Visit 2 or 3, uterine biopsy or ovariohysterectomy was performed for histology. Of nine eligible queens, seven were non-pregnant by ultrasound at Visit 2 and two had lost pregnancies by Visit 3. Ovulation was confirmed by serum progesterone concentration in all queens. Ultrasonic appearance of the ovaries and uterus was compatible with a healthy status except for one queen with signs of cystic endometrial hyperplasia (CEH) and pyometra, a follicular cyst in another, and fetal resorptions in two queens. Six cats had histologic lesions of endometrial hyperplasia, including CEH (n=1). Only one cat had no histologic uterine lesions. Bacteria were cultured from vaginal samples in seven queens at Visit 1, (two were non-evaluable), and in five of seven queens sampled at Visit 2. Uterine cultures were negative except for the cat with pyometra. All urine cultures were negative. In summary, the most frequent pathology observed in these infertile queens was histologic endometrial hyperplasia, which can potentially inhibit embryo implantation and healthy placental development. This suggests that uterine disease might contribute substantially to infertility in purebred queens.

Differential expression of inflammatory cytokines, prostaglandin synthases and secretory leukocyte protease inhibitor in the endometrium and circulation in different graded CEH-pyometra in bitch.

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3-10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5-2.23 fold higher in mCEH-P, 0.87-2.5 fold higher in sCEH-P and 2.9-3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness.