Uterine Biopsies Research Articles

Changes in expression pattern of selected endometrial proteins following mesenchymal stem cells infusion in mares with endometrosis.

Mesenchymal stem cells (MSCs) due to their self-renewal potential and differentiation capacity are useful for tissue regeneration. Immunomodulatory and trophic properties of MSCs were demonstrated suggesting their use as medicinal signaling cells able to positively change local environment in injured tissue. Equine endometrosis is a progressive degenerative disease responsible for glandular alterations and endometrial fibrosis which causes infertility in mares. More precisely, this disease is characterized by phenotypic changes in the expression pattern of selected endometrial proteins. Currently, no effective treatment is available for endometrosis. Herein, we aimed at the evaluation of expression pattern of these proteins after allogeneic equine adipose tissue-derived multipotent mesenchymal stem cells (eAT-MSCs) infusion as well as at testing the capacity of these cells to promote endometrial tissue remodeling in mares with endometrosis. eAT-MSC (2×107/animal) were transplanted into mares’ uterus and control animals received only placebo. Uterine biopsies were collected before (day 0) and after (days 7, 21 and 60) cells transplantation. Conventional histopathology as well as expression analysis of such proteins as laminin, vimentin, Ki-67-antigen, α-smooth muscle actin (α-SMA) and cytokeratin 18 (CK18) have been performed before and after eAT-MSCs transplantation. We demonstrated that eAT-MSCs induced early (at day 7) remodeling of endometrial tissue microenvironment through changes observed in intra cellular and intra glandular localization of aforementioned proteins. We demonstrated that eAT-MSCs were able to positively modulate the expression pattern of studied secretory proteins as well as, to promote the induction of glandular epithelial cells proliferation suggesting local benefits to committed endometrial tissue environment after eAT-MSCs transplantation.

P16INK4a and Ki67 expression in normal, dysplastic and neoplastic uterine cervical epithelium and human papillomavirus (HPV) infection

Cellular cycle proteins like the p16INK4a and the Ki67 proliferation nuclear antigen have been used as oncogenicity cellular markers. The E6 and E7 oncoproteins interact with tumor suppressor genes p53 and pRb, culminating with the p16INK4a overexpression.The objective of this study was to evaluate the presence of HPV-DNA in 174 cervical biopsies and correlate the different histological grades with the p16INK4a and Ki67 immunohistochemical expression (IHC).A cross-sectional study that enrolled a total of 174 women who underwent uterine cervical biopsies between February 2003 and December 2006, in southern Brazil, was performed. Cervical smear samples were analyzed for the presence of HPV-DNA through polymerase chain reaction (PCR), and biopsy samples were examined for p16INK4A and Ki67 expression through IHC techniques.The presence of HPV-DNA was observed in 89% of the tested patients, among which 52% were positive for high-risk (HR) viral types [16, 18 and 31]. Regarding p16INK4a, an expression of 69% was observed, being expressed in 100% of the high-grade squamous lesions (HSIL) and HR-HPV-DNA positives. Ki67 expression was associated with the lesion grade, being more expressive in the most severe lesions (p<0.001). p16INK4A and Ki67 markers coexpression was present in 86% of the samples (p<0.001), being 100% among those positive to HR-HPV-DNA with HSIL (p<0.001).The results suggest an association between the presence of HR-HPV infection and the p16INK4a and Ki67 expression and which is even stronger among women with HSIL.

Fourier-domain Jones-matrix mapping of a complex degree of mutual anisotropy in differentiation of biological tissues' pathological states.

This article presents the theoretical background of an azimuthally stable method of Jones-matrix mapping of histological sections of a uterine wall biopsy on the basis of spatial-frequency selection of the mechanisms of linear and circular birefringence. The diagnostic application of a new correlation parameter--a complex degree of mutual anisotropy--is analytically substantiated. The method of measuring coordinate distributions of a complex degree of mutual anisotropy with further spatial filtration of their high- and low-frequency components is developed. The interconnections of such distributions with linear and circular birefringence parameters of the uterine-wall-endometrium histological sections are found. The comparative results of measuring the coordinate distributions of a complex degree of mutual anisotropy formed by fibrillar networks of myosin and collagen fibrils of uterus wall tissue of different pathological states--pre-cancer (dysplasia) and cancer (adenocarcinoma)--are shown. The values and ranges of change of the statistical (moments of the first to fourth orders) parameters of complex degree of mutual-anisotropy coordinate distributions are studied. The objective criteria of diagnosing the pathology and differentiation of its severity degree are determined.

Histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (1990–2005)

To determine the prevalence of various lesion types detected by histologic evaluation of uterine biopsy samples collected from subfertile bitches. Retrospective case series. 399 sexually intact bitches. Results of histologic evaluation of canine uterine biopsy samples submitted by a single veterinary practice and clinical histories of dogs from which samples were obtained were reviewed. Clinical data including age, reason for biopsy, and histopathologic findings were recorded. The prevalence of specific lesions was determined, categorized by severity and age, and statistically analyzed. Endometritis (170/399 [42.6%] cases) and cystic endometrial changes, including cystic endometrial hyperplasia (133/399 [33.3%]) were the most prevalent lesions in the study population. Eighty-nine of 170 (52.4%) cases of endometritis were characterized as chronic with predominantly lymphocytic or lymphoplasmacytic inflammatory infiltrates, 51 (30.0%) included mixed inflammatory reactions, and 30 (17.6%) were characterized as having acute inflammation with neutrophils, eosinophils, or both. Fibrosis was common (101/399 [25.3%] cases). Eosinophilic endometritis was significantly associated with a history of fetal loss during the same breeding cycle. No significant difference was found in prevalence of lesions among age groups. The high prevalence of endometritis in this population of dogs suggested that acute and chronic endometritis may be related to subfertility in bitches. The association of eosinophilic endometrial infiltrates with a history of fetal loss may be an important diagnostic finding in dogs with endometritis.

Assessment of myometrial transcriptome changes associated with spontaneous human labour by high‐throughput RNA‐seq

The transition of the human uterus from a quiescent to a contractile state takes place over a number of weeks. On such biological time scales, cellular phenotype is modified by changes in the transcriptome, which in turn is under the control of the underlying endocrine, paracrine, and biophysical processes resulting from the ongoing pregnancy. In this study, we characterize the transition of the human myometrial transcriptome at term from not in labour (NIL) to in labour (LAB) using high throughput RNA sequencing (RNA-seq). RNA was isolated from the myometrium of uterine biopsies from patients at term who were not in labour (n=5) and at term in spontaneous labour (n=5) without augmentation. A total of 143.6million separate reads were sequenced, achieving, on average, ∼13timescoverage of the expressed human transcriptome per sample. Principal component analysis indicated that the NIL and LAB transcriptomes could be distinguished as two distinct clusters. A comparison of the NIL and LAB groups, using three different statistical approaches (baySeq, edgeR, and DESeq), demonstrated an overlap of 764 differentially expressed genes. A comparison with currently available microarray data revealed only a partial overlap in differentially expressed genes. We conclude that the described RNA-seq data sets represent the first fully annotated catalogue of expressed mRNAs in human myometrium. When considered together, the full expression repertoire and the differentially expressed gene sets should provide an excellent resource for formulating new hypotheses of physiological function, as well as the discovery of novel therapeutic targets.

Development and Comparison of Sampling Techniques for a Broad Range, Semiquantitative Polymerase Chain Reaction Assay for Detection of Bacterial DNA in the Equine Uterus

Microbial culture from a double-guarded culture swab is commonly used to diagnose infectious endometritis. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR) assay to detect a broad range of bacteria from equine uterine samples. Twenty-seven mares with a clinical history of endometritis had a double-guarded culture swab collected for analysis by qPCR and microbial cultures. An additional 12 mares had a uterine biopsy sample collected for qPCR analysis, microbial culture, and histopathology. Subsequently, a double-guarded culture swab for microbial culture and a cytology brush sample were also collected. The qPCR assay detected bacterial DNA in nine of 27 mares from a double-guarded swab and six of 12 mares from an endometrial biopsy. Positive microbial growth was detected in nine of 27 mares and four of 12 mares from a double-guarded culture swab. Bacterial DNA was detected in two of 27 mares and two of 12 mares without subsequent microbial growth. The simple presence of an organism's DNA allows for detection by nonculture-based systems, both live and dead organisms can be identified. In conclusion, the qPCR assay was determined to be a sensitive diagnostic technique for identifying pathogens associated with infectious endometritis. The primary application of the qPCR assay is detection of potential pathogenic bacteria in the uterus of a mare suspected of having infectious endometritis when a traditional microbial culture is negative. Further work is warranted to determine if mares positive for bacterial DNA and negative for microbial culture are affected clinically.

The utility of p16INK4A expression in uterine cervical biopsies reported negative and positive for dysplasia at Kenyatta National Hospital, Nairobi, Kenya

Background Hematoxylin and eosin (HE mean age: 40.2 years. On review, negative for dysplasia were 32, CIN1 19, CIN2 16 and CINIII 24 (p Conclusion There is significant inter-observer variation in interpretation of cervical biopsies on H&E. p16INK4a expression correlates well with high grade lesions.

Preoperative diagnosis of usual leiomyoma, atypical leiomyoma, and leiomyosarcoma.

Uterine smooth muscle tumors (SMTs) are common pelvic tumors in women, and most of them are diagnosed as usual leiomyoma (UL). Exclusion of malignant disease is important in the management of SMTs. However, differentiation of SMTs remains difficult. In this study, we aimed to improve the preoperative diagnosis of SMTs. We examined 21 ULs, 7 atypical leiomyomas (ALs), and 6 leiomyosarcomas (LMSs), all of which were diagnosed by uterine tumor biopsy. Immunohistochemical findings (low-molecular-mass polypeptide 2 (LMP2) and Ki-67) and clinical features (serum lactate dehydrogenase level and menopause) were evaluated. Statistically significant differences in the expression of LMP2 and Ki-67 were observed between UL and AL and between UL and LMS. The combined LMP2 and Ki-67 score was significantly different between UL and AL, between UL and LMS, and between AL and LMS. The combined immunohistochemistry and clinical findings score (total score) was also significantly different between pathological types. The findings of this study suggest that the accuracy of the preoperative diagnosis of SMTs may be improved by using a combination of immunohistochemical and clinical findings.

The effects of intra-uterine kerosene infusion on endometrial epithelial cilia concentration

The endometrial muco-ciliary apparatus plays an important role in the clearance of fluid and inflammatory debris from the uterus. It has been documented that there is a significant difference in the distribution of ciliated cells between mares with normal fertility and mares with delayed uterine clearance. The infusion of kerosene into the uterus as a treatment for chronic infertility in mares has been described. Endoscopic, histologic and cytologic examination post infusion reveals severe acute inflammation that gradually resolves over seven to fourteen days. The goal of this study was to determine if the intra-uterine infusion of kerosene had an effect on the concentration of ciliated endometrial epithelial cells detectable via histologic examination of endometrial biopsies. Uterine biopsy samples were obtained from nine reproductively healthy mares the cycle prior to treatment. Four mares served as controls and were infused with saline, and five mares were treated with intra-uterine infusion of kerosene. Two hundred and fifty milliliters of saline or kerosene were infused into the uterus during estrus and uterine biopsy samples were acquired one, four, seven and twenty-one days after administration. The biopsy samples were fixed in Bouin’s solution and standard H&E sections were prepared. The percentage of ciliated endometrial epithelial cells was determined by counting a minimum of 100 endometrial cells and assessing the percentage that possessed tufts of tall cilia. There was no statistical difference between the

Upregulation of TLR-4 and proinflammatory cytokine transcripts as diagnostic indicator of endometritis in buffaloes

The aim of present study was to examine the relative mRNA transcription of toll-like receptor 4 (TLR-4), CD14, interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) in endometritic buffaloes. Reproductive tracts were collected from 60 buffaloes at postmortem. The genitalia were diagnosed for endometritis based on positive white side test of uterine fluid, presence of >5% polymorphonuclear cells in uterine cytology, and endometrial histopathology. The endometritis-positive genitalia were categorized as luteal (n = 10) and follicular (n = 10) based on the presence of corpus luteum. Further, 20 normal genitalia (luteal, n = 10 and follicular, n = 10) were also included as control. The endometrial tissue scrapings were collected ex vivo from all the samples, total RNA was extracted, complementary DNA was transcribed for each sample, and relative quantification of mRNA was done by real-time polymerase chain reaction. The threshold cycle values of all the samples were normalized with the housekeeping β-actin gene and relative fold change was calculated using 2−ΔΔCt method. Present study revealed a variable upregulation of TLR-4, CD14, IL-1β, IL-8, and TNF-α transcription in endometritic buffaloes either at luteal or follicular stage. Predominantly at luteal stage, the significant upregulation of TLR-4, IL-8, and TNF-α transcripts was 4.66-, 21.19-, and 42.49-fold, respectively, indicated the possibility of cytokine-based diagnostic method for the early indication of endometritis before the onset of cyclic estrus. Nevertheless, further research in respect to threshold level of each cytokine in uterine biopsies involving more number of animals is required to establish a potential marker gene(s) for the diagnosis of endometritis and for devising novel therapeutic intervention.

184 ISOLATION AND CHARACTERIZATION OF BOVINE ENDOMETRIAL STEM CELLS

Adult mesenchymal stem cells had been isolated from various tissues of different species, including endometrial tissue of humans, mice, and pigs, but not from cattle. The aim of our work was to identify such cells in the bovine endometrium and to establish a model system in which to test inducers of differentiation and recruiters of stem cell niches, for potential therapeutic use in this and other species, such as horses. We searched for endometrial stem cells in healthy cycling cows and in cattle with clinical (C) or subclinical (SC) endometritis. For this, the uterine tracts of slaughtered cows were collected at early (Days 2 to 5; ELF) and late luteal phases (Days 11 to 15; LLF) of the oestrus cycle of healthy cows. For endometritis-diseased cattle, uterine biopsies were taken in live animals. In all cases, markers of stemness, inflammation, uterine function, and housekeeping were studied both at mRNA and protein level, by RT-qPCR and Western blot/immunohistochemistry respectively. In addition, cell primary cultures were established in vitro from all the animals (n = 4 for ELF, n = 4 for LLF; n = 4 for C and n = 4 for SC). We found that the endometrium of the majority of studied animals expressed embryonic stem cell markers, OCT4 and SOX2, but not or little NANOG, as well as CD44, c-Kit, and STAT3, all markers of mesenchymal stem cells. The expression profile of these markers was not related to the stage of the oestrus cycle; however there was a statistically significant reduction in the expression of embryonic stem cell markers in ill animals, being the lowest in clinically ill and intermediate in subclinical endometritis, (P &lt; 0.05 and Pearson correlation coefficient 0.92). For markers of multipotency (mesenchymal), the expression was lower in clinical endometritis (P &lt; 0.05). In conclusion, the expression profile of stem cell markers is indicative of the presence of stem cells in the bovine endometrium. At the protein level, we verified our findings for OCT4, SOX2, and CD44 using Western blot and immunohistochemistry. In general, there was a concordance between mRNA and protein profiles. Inflammatory markers showed a pattern characteristic for each of the studied stages. In order to have an ultimate criterion of the presence of stem cells, we tested the differentiation potential of the isolated cell lines, upon induction towards chondrogenic, osteogenic, and adipogenic lineages. We found that all the cell lines tested (n = 8) displayed mesenchymal differentiation potential as demonstrated by specific staining and gene expression markers. At present, work is in progress to isolate pure stem cell populations from these primary cultures to further characterise these cells. Conclusion: we showed for the first time the presence and differentiation potential of endometrial stem cells in cattle. This can have an effect on the development of new therapeutic approaches to combat uterine diseases, such as endometritis or endometriosis (in horses). This work was supported by grant FONDECYT REGULAR 1110642, from the Government of Chile.

Prevalence of subclinical endometritis in repeat breeding cows and mRNA expression of tumor necrosis factor α and inducible nitric oxide synthase in the endometrium of repeat breeding cows with and without subclinical endometritis

Information on the prevalence of subclinical endometritis and its mechanism in repeat breeding cows is very limited. The aims of this study were: a) to evaluate the incidence of this disorder with cytobrush cytology b) to analyze mRNA expression of tumor necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) in endometrial biopsy samples collected from repeat breeding cows with and without subclinical endometritis. Two experiments were carried out. In experiment 1,112 (12.4%) repeat breeding cows (inseminated at least 3 times and not pregnant) were selected out of 902 cows from 8 dairy herds. Cytobrush cytology was performed on these cows, using the threshold of 10% PMNs in uterine smears. The results showed that 45 out of the 112 cows (40.2%) were diagnosed as having subclinical endometritis. In experiment 2, uterine biopsy samples were taken from repeat breeding cows with subclinical endometritis (n = 10) and without this disorder (n = 10). Using reverse transcription-PCR, the mRNA expression of TNFalpha and iNOS was determined. A statistically significant increase in expression of both substances was measured in the group of cows with subclinical endometritis (p <0.05). These results provide evidence for a high prevalence of subclinical endometritis in repeat breeding cows as well as the involvement of TNFalpha and iNOS pathways in the regulation of this pathological condition.

Endometrial cytology, biopsy, and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows

The objectives of this study were to assess the agreement between endometrial cytology and uterine biopsy for the diagnosis of subclinical endometritis (SEND) in grazing dairy cows, the interobserver agreement of the biopsy’s readings, and the bacterial population isolated from the uterus of cows having SEND. In experiment 1, lactating Holstein cows (n=44) 31 to 59d in milk (DIM) at sampling were enrolled. Clinical endometritis was diagnosed by direct evaluation of vaginal discharge and SEND by endometrial cytology evaluation. Two hundred cells per smear were counted to determine the percentage of polymorphonuclear neutrophilic leukocytes (PMNL). Cut-off values used were ≥8% PMNL at ≤33 DIM, ≥6% PMNL at 34 to 47 DIM, and ≥4% PMNL at ≥48 DIM. Biopsies were assessed blindly by 2 observers who categorized them into 4 groups according to their inflammatory changes: none, minimal, moderate, and severe inflammatory changes. Data were analyzed using the kappa coefficient and logistic regression. In experiment 2, lactating Holstein cows (n=60) 21 to 62 DIM were enrolled. Clinical endometritis and SEND were diagnosed as previously described. Samples were cultured for aerobic and anaerobic bacteria by routine methods of bacteriological testing. Data were analyzed with logistic regression. In experiment 1, little agreement was observed between cytology and biopsy outputs (kappa=0.151), and strong agreement between the 2 operators (kappa=0.854). The likelihood of having a normal biopsy (no inflammatory change) was greater for healthy cows than for those having SEND (odds ratio=13.145). The probability for getting normal uterine tissue decreased 2.1% for every increasing percentage point in PMNL. In experiment 2, no bacteria were isolated from cows with SEND, coagulase-negative staphylococci were commonly isolated from healthy cows, and Trueperella pyogenes was frequently isolated from cows with clinical endometritis. The likelihood of isolating T. pyogenes from uterine samples increased with the percentage of PMNL (odds ratio=1.100). In conclusion, biopsy showed low agreement with cytology for the diagnosis of SEND. Nevertheless, fertility trials using uterine biopsies to predict pregnancy outcomes are needed to determine its diagnostic usefulness. Finally, bacteriology would not be recommended as a diagnostic tool because no bacteria were isolated from cows with SEND.

HISTOPATHOLOGICAL ECTOCERVIX EPITHELIUM CHANGES IN PATIENTS WITH HPV-INFECTION

Aim. To study the histopathological changes in ectocervix epithelium among patients with HPV-infection. Materials and methods. The object of the study – 105 women aged 25 to 39 with PCR-diagnosed HPV-infection. Patients underwent standard gynecological investigation and widened colposcopy with a target (colposcopically oriented) uterine cervix biopsy. Biopsy material was subjected to histological investigation using the generally accepted histological methods. Statistical analysis was performed with the program package Biostat. For the purpose of assessing significance of differences between distribution of qualitative signs in the studied samples, χ 2 criterion and Z criterion were used. Results. Patients with HPV-infection had some morphological variants of changes in uterine cervix mucosa that depends on presence and degree of multilayer pavement epithelium dysplasia. It was established that while CIN is progressing, expression of such HPV-infection signs as manifestation of koilocytosis and hyper-and parakeratosis is reduced. Besides, there is a tendency to chronization of inflammatory changes in uterine cervix lamina as CIN degree is growing. As CIN degree is growing, there is increase in cervical glandular intraepithelial neoplasia degree (CGIN). Conclusion. As a result of histological investigation of biopsy material taken from HPV-infected patients with uterine cervix pathology, regularities of changes in the character of reactive ectocervix processes connected with CIN progressing were established.

Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003–2008)

To describe bacteria isolated from reproductive tracts of mares and to examine the extent and patterns of resistance to antimicrobials commonly used for treatment of endometritis. Retrospective case series. 8,296 uterine swab, lavage, or biopsy samples obtained between January 2003 and December 2008 from 7,665 horses in central Florida. Results of bacterial culture and antimicrobial susceptibility testing were obtained for uterine swab, lavage, and biopsy samples collected from mares undergoing a routine breeding examination or examined because of a reproductive disorder. Bacterial organisms were identified by means of standard techniques, and proportions of samples resistant to various antimicrobials were determined. At least 95% of samples (n = 1,451) were collected with uterine swabs. Potentially pathogenic organisms were cultured from 2,576 (31%) samples, with Escherichia coli (n = 729 [29%]) and β-hemolytic Streptococcus equi subsp zooepidemicus (733 [28%]) being most common. Resistance to antimicrobials changed over time for E coli, S equi subsp zooepidemicus, and Enterobacteriaceae isolates. Overall, E coli was most resistant to trimethoprim-sulfonamide and ampicillin and least to amikacin and enrofloxacin. For S equi subsp zooepidemicus, resistance was greatest to oxytetracycline and enrofloxacin and least to ceftiofur and ticarcillin with or without clavulanic acid. Inflammatory response was greater for S equi subsp zooepidemicus. E coli and S equi subsp zooepidemicus were the most common pathogens recovered from uterine samples, with S equi subsp zooepidemicus more commonly associated with inflammation. Antimicrobials most commonly used empirically to treat endometritis are appropriate on the basis of these data. However, as antimicrobial resistance changes over time, susceptibility assays should aid antimicrobial selection.

A novel strategy of mesenchymal stem cells delivery in the uterus of mares with endometrosis

Mesenchymal stem cells (MSCs), because of their immunomodulation and trophic activities, in addition to their capacity to regenerate damaged tissues, have potential for treatment of many diseases. The success of stem cell therapies depends, in part, on the method of cell delivery, which should provide wide cell distribution and homing in to injured sites. The objective of the present study was to developing a novel strategy for delivery of MSCs into the uterus of mares with endometrosis (degenerative alteration of uterine glands and surrounding stroma). Endometrosis was confirmed in all mares (N = 6) used in this study. To trace multipotent equine adipose tissue-derived MSCs (eAT-MSCs) in endometrial tissue, before transplantation, cells were stained with a fluorescent dye. During a synchronized estrus, the eAT-MSCs (2 × 107 diluted in 20 mL of sodium chloride 0.9%) were inoculated into uterus using a simple technique, similar to artificial insemination (AI) in mares. At 7 and 21 days after transplantation, homing of fluorescently labeled eAT-MSCs was observed by confocal microscopy of uterine biopsies collected from the uterine body and in both uterine horns, including glandular and periglandular spaces, in three of four treated mares. Herein, we propose a new method of MSCs delivery in uterus of mares with endometrosis, which was minimally invasive and technically simple.

Chronic levothyroxine and acute T3 treatments enhance the amplitude and time course of uterine contractions in human

This study compares the functional consequences of levothyroxine (T4) treatment during pregnancy as well as the acute affects of triiodothyronine (T3) on spontaneous uterine contractile activities observed in vitro. Uterine biopsies were obtained from consenting women undergoing elective caesarean at term (n = 28). Spontaneous contractile activities from T4-treated pregnant women (n = 8) were compared with control patients (n = 20) by isometric tension measurements. Effects of acute T3 and T4 on control tissues were also monitored. Area under the curve, amplitude, time to peak, duration, and frequency were quantified. In uterine strips from women treated for hypothyroidism, phasic uterine contractions of larger amplitude (+77%) were observed, with a prolonged duration at 90% relaxation (+138%) and reduced frequency (-55%) compared with values of the control group. The addition of exogenous T3 in vitro on control strips induced a significant increase in the duration of the contractions and a significant decrease in frequency (P < 0.05), which partially mimics the results obtained in strips from T4-treated women. Significant modifications of contractile properties were observed in strips from pregnant women treated with levothyroxine, consistent with those observed with the addition of exogenous T3. Clinical practices of modern obstetrics should take into account the effect of thyroid hormones on uterine contractions' time course to ensure a tighter followup at the end of pregnancy to achieve safer delivery.

An Investigation of Uterine Nitric Oxide Production in Mares Susceptible and Resistant to Persistent Breeding‐Induced Endometritis and the Effects of Immunomodulation

The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) within 24h after breeding. Mares susceptible (n=6) or resistant (n=6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexamethasone or mycobacterial cell wall extract (MCWE) on uterine NO production and endometrial iNOS mRNA expression. Six susceptible mares were inseminated over three cycles with (i) killed spermatozoa without treatment (control), (ii) killed spermatozoa with 50mg of dexamethasone IV or (iii) MCWE IV 24h prior to insemination with killed spermatozoa. Six resistant mares were inseminated with killed spermatozoa as a control. Six hours after breeding, uterine biopsies and secretions were collected and evaluated for NO and iNOS mRNA. In Experiment 1, resistant mares had an increase in iNOS mRNA expression 2h post-breeding compared to baseline (p=0.045), 12h (p=0.014) and 24h (p=0.001). Susceptible mares had higher expression 2h compared to 6h (p=0.046). No differences were observed in mRNA or protein expression of iNOS between resistant and susceptible mares. Resistant mares had a relatively steady amount of total intrauterine NO over 24h, while susceptible mares had an increase over time, with a significantly higher increase in total NO than resistant mares at 6 (p=0.04) and 12h (p=0.032). In Experiment 2, no differences were observed for iNOS mRNA expression. Susceptible mares had increased NO when compared to resistant mares (p=0.008) and MCWE decreased NO (p=0.047).

96 EXPRESSION OF THE LONG FORM OF THE LEPTIN RECEPTOR IN BOVINE OVIDUCT EPITHELIAL, UTERINE EPITHELIAL, AND BLASTOCYST-STAGE EMBRYOS

Critical reproductive events of animals are affected via metabolic cues that directly modulate the hypothalamic-pituitary-gonadal axis. The adipose-derived 146-amino-acid protein-hormone leptin has been associated with puberty and reproduction in many mammals and may be of critical importance in the regulation of reproduction. Leptin receptors are expressed in the brain, pituitary, and gonads, and there is growing evidence that leptin may be required for normal embryonic implantation and development in some animals. To date 6 isoforms of the leptin receptor have been identified; the long form (Ob-Rb) is the only isoform to be associated with signal transduction. The objective of this experiment was to test the hypothesis that the Ob-Rb isoform is expressed in cultured bovine oviduct and uterine epithelial cells, in vivo-derived mid-luteal uterine biopsies, and expanded bovine blastocyst-stage embryos. For cells, cell lines from mid-luteal abattoir-derived reproductive tracts (n = 5) were used; for uterine biopsies (n = 20), biopsies were collected from both uterine horns at the greater curvature from mature beef cows (n = 4) in mid-luteal phase; expanded IVP blastocyst-stage embryos (n = 40) were produced from abattoir-derived ovaries. For fluorescence microscopy samples were incubated in rabbit anti-human leptin receptor (#4781-L, Linco Research, St. Charles, MO) followed by Alexa Fluor 488 anti-rabbit IgG (#A-11008, Molecular Probes, Carlsbad, CA). We confirmed that the Ob-Rb isoform is expressed in both oviductal and uterine in vitro-cultured epithelial cells and uterine biopsies. All oviduct but not all uterine-cultured epithelial cells were immunopositive for the Ob-Rb isoform. All uterine biopsies were positive for the Ob-Rb isoform along the luminal side of the uterine endometrium with an apparent lack of staining occurring in myometrial tissue. Moreover, we demonstrated that all bovine blastocysts were immunopositive with staining occurring within the trophoblast cell type but not the inner cell mass. These results show for the first time that bovine oviduct and uterine epithelial cells express the long form of the leptin receptor and support earlier findings that blastocyst-stage embryos contain Ob-Rb isoforms. This suggests that the Ob-Rb isoform is a component of the bovine maternal-embryonic complex; however, its function in pre-implantation embryo development remains to be clarified.

Diseases of the Canine Uterus

Lesions most commonly found in the canine and feline uteri fall primarily into two categories: (i) abnormalities of endometrial growth and repair and (ii) uterine infections with associated endometritis. Neoplastic conditions of the tubular genitalia of the bitch, with the possible exception of leiomyomas (tumours that arise from smooth muscle cells), are uncommon. Congenital lesions involving the uterus are relatively rare. The primary object of this paper is to provide a summary of the most common uterine lesions found in the canine and feline uterus using a series of images that underscore the most prominent and important gross diagnostic features of each.