Influence of cycle stage, age and endometrial biopsy score on oxytocin receptor distribution and gene expression in the cervix and uterus of non-pregnant mares.

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined. In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2-9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

[A case of 63,X/64,XX mosaicism in a subfertile pony mare]. / Ein Fall von 63,X/64,XX Mosaizismus bei einer subfertilen Ponystute.

INTRODUCTION: The present case report describes a 6-year old subfertile pony mare, which became pregnant after the eleventh artificial insemination. The examination of the ovaries and the uterus did not reveal any abnormal clinical findings and the mare showed a regular oestrous cycle. Based on cytogenetic and molecular genetic analyses it became possible to elucidate the observed subfertility. The mosaic karyotype of the mare consisted of 63,X (20%) and 64,XX (80%) cells. A PCR analysis failed to amplify sequences from the equine SRY gene. The observed classic 63,X/64,XX mosaicism is a plausible explanation for the subfertility of the mare.

Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa.

Preservation of epididymal spermatozoa is important to save genetic material of endangered species and breeds, or in case of unexpected injury, which will end the breeding career of valuable sires. Seminal plasma (SP) influences sperm quality in a dose-dependent manner and its addition to preserved semen immediately before insemination may be beneficial for sperm fertility. Increased plasma membrane stability of epididymal spermatozoa reduces freezing injury of cells, and the addition of SP after freezing and thawing might have activating and protecting effects on spermatozoa within the female genital tract. In this study, epididymal spermatozoa were harvested by retrograde flush of the epididymal cauda immediately after routine castration and frozen. Seminal plasma was collected from other six stallions. Homologous SP (SP from the same species, but from a different animal) was added to frozen-thawed epididymal spermatozoa at concentrations of 0, 5, 20, 50 and 80% SP. Addition of SP increased sperm motility and influenced kinematic values in a dose-dependent manner (p < 0.05). Motility improved at concentrations of 20 and 50% SP, but did not further increase at 80% SP. There was no difference in sperm motility among SP from six different donor stallions regardless of the concentrations of SP (p > 0.05). Total and progressive motility of ten frozen-thawed epididymal spermatozoa samples collected from different stallions after dilution with extender and 5, 20, 50 or 80% SP differed significantly (p < 0.05). In conclusion, addition of homologous SP to frozen-thawed stallion epididymal spermatozoa immediately improved motility in a dose-dependent manner regardless of semen quality of SP donor stallions. This might positively influence fertility when SP is added before insemination. Moreover, there seems to be a threshold level of SP concentration for optimal improvement of sperm motility.

[Clinical relevant procedures for early pregnancy diagnosis in the mare]. / Klinisch relevante Vorgehensweisen zur frühzeitigen Überprüfung der Konzeption bei der Stute.

This review describes stepwise the recto-manual and transrectal ultrasonographic evidence of early pregnancy detection in the horse. The morphological and physiological conditions in the individual phases of early pregnancy are presented in correlation to the potential clinical findings. The importance of embryonic and early foetal losses is presented. Communication and documentation of findings are also addressed. The final section is devoted to the evaluation of the examination effort. In this regard, it is emphasized that the gynaecological examination for the evaluation of the pregnancy status represents a service contract.

Colour analysis of the equine endometrium: comparison of spectrophotometry and computer-assisted analysis of photographs within the L*a*b* colour space system.

The aims of this study were to compare two different methods of quantifying the colour of the luminal surface of the equine endometrium and to relate the results to histopathological evidence of inflammation and fibrosis. The mucosal surfaces of 17 equine uteri obtained from an abattoir were assessed using a spectrophotometer and by computer-assisted analysis of photographs. Values were converted into L(*)a(*)b(*) colour space. Although there was significant correlation between the two methods of quantification, variations in 'brightness', 'red' and 'yellow' values were noted. Within a given uterus, measurements using the spectrophotometer did not differ significantly. Using photographic analysis, brightness differed between horns, although no differences in chromaticity were found. Histopathological classification of changes within endometria corresponded to measured differences in colour. Extensive fibrosis was associated with increased brightness and decreased chromaticity using both methods. Inflammation correlated with reduced chromaticity, when measured by spectrophotometry, and with reduced brightness and yellow values, when assessed photographically. For this technique to gain wider acceptance as a diagnostic tool, e.g. for the endoscopic evaluation of uterine mucosae in vivo, standardised illumination techniques will be required so that colours can be compared and interpreted accurately.

Factors influencing foaling rate following colic surgery in pregnant Thoroughbred mares in Central Kentucky.

REASONS FOR PERFORMING STUDY: Clinical experience has suggested that foaling rates following colic surgery in the pregnant mare are influenced by days of gestation. This premise has not been supported in previous studies. We also aimed to determine the effects of other potential influencing factors. OBJECTIVE: To determine the influence of age of gestation on foaling rates in pregnant mares following colic surgery and evaluate the relationships between other factors and foaling rates. METHODS: Medical records of Thoroughbred mares, which had colic surgery performed from 1993 to 2007 at the Rood and Riddle Equine Hospital, were reviewed. Mares identified in the record as pregnant or bred within the previous 15 days were included in this study. Age of the mare, date of surgery, gestational age, duration of colic at admission, packed cell volume at admission, surgical diagnosis, duration of general anaesthesia, intraoperative hypotension, intraoperative hypoxaemia and post operative signs of endotoxaemia were recorded. A mare was considered to have a live foal if that foal was registered with the North American Jockey Club. RESULTS: Of the 228 mares, where pregnancy had been confirmed, 152 (66.7%) had a live foal registered after surgery. Mares bred <40 days before surgery had a lower foaling rate compared with mares undergoing surgery ≥40 days after breeding: 48.7% vs. 69.8% (odds ratio [OR] = 0.41 [95% confidence interval (CI) 0.20-0.83], P = 0.012). Foaling rate was also influenced by mare's age (P = 0.008) and duration of colic signs before surgery (P = 0.03). CONCLUSIONS: The prognosis for a live foal after colic surgery in the pregnant Thoroughbred mare is significantly better if the mare is ≤15 years of age and ≥40 days of gestation. POTENTIAL RELEVANCE: The results of this study are useful for clinicians offering a prognosis for a live foal following colic surgery in pregnant mares.

Microarray analysis of equine endometrium at days 8 and 12 of pregnancy.

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Diseases and medical disabilities of enslaved Barbadians, from the seventeenth century to around 1838 part II.

The disease environment, health problems and causes of mortality of enslaved Barbadians are described. Data are derived mainly from documentary sources; also included are bio-archaeological data from analyses of skeletons recovered from Newton Plantation cemetery. Major topics include infectious diseases transmitted from person to person, as well as those contracted through water soil, and other environmental contaminations, and diseases transmitted by insects, parasites and other animals; nutritional diseases, including protein energy malnutrition, vitamin deficiencies, anaemia, and geophagy or "dirt eating"; dental pathologies, lead poisoning, alcoholism, traumas, and other disorders, including psychogenic death or illness caused by beliefs in witchcraft or sorcery.

A three-dimensional culture model of canine uterine glands.

The canine endometrium is frequently affected by severe alterations with unclear pathogenesis and is, therefore, an important subject of research in veterinary gynecology. Therefore, the aim of our study was to establish a three-dimensional in vitro system of the canine endometrium suitable for experimental approaches. For this reason, intact uterine glands were isolated from canine uteri and placed together with stromal cells on culture dishes coated with several extracellular matrix components (collagen I, IV, fibronectin, laminin, gelatin, Matrigel) for up to 4 d to support differentiation of cultured cells. Immunohistochemical detection of laminin on freshly isolated glands showed a partial preservation of the basement membrane--an important factor for epithelial differentiation. Glandular structures were differentiated and polarized during culture time as shown by electron microscopy. Signs of degeneration and loss of cell-cell adhesions as seen occasionally on day 4 depended on the individual dog. In general, morphology was best preserved on Matrigel matrix. No significant changes of cultured glandular explants were observed concerning proliferation and steroid receptor (estrogen, progesterone) expression when compared with the original uterine tissue as assessed by immunohistochemical staining. Lectin histochemistry revealed comparable results for the in vivo endometrial glands and the cultured glandular explants during the whole culture period. This in vitro reconstitution of the canine endometrium is a promising tool to study the cyclic events in the normal endometrium as well as alterations in the affected uterus.

Diseases and medical disabilities of enslaved Barbadians from the seventeenth century to around 1838. Part I.

The disease environment, health problems and causes of mortality of enslaved Barbadians are described. Data are derived mainly from documentary sources; also included are bio-archaeological data from analyses of skeletons recovered from Newton Plantation cemetery. Major topics include infectious diseases transmitted from person to person, as well as those contracted through water soil, and other environmental contaminations, and diseases transmitted by insects, parasites, and other animals; nutritional diseases, including protein energy malnutrition, vitamin deficiencies, anaemia, and geophagy or "dirt eating"; dental pathologies; and lead poisoning, alcoholism, traumas, and other disorders, including psychogenic death or illness caused by beliefs in witchcraft or sorcery.

Cognitive effects of short-term use of raloxifene: a randomized clinical trial.

Two questions regarding findings from the Women's Health Initiative are (1) What is the effect of various hormonal regimens including selective estrogen receptor modulators? and (2) Is the negative effect on cognitive functioning related to the older age (65+ years) of the women? This study addresses these two questions in a short-term randomized trial of the effects of raloxifene versus alendronate on cognition. The study found only one significant interaction where the raloxifene and alendronate group changed differently across the two testing occasions. Hence, raloxifene does not have any impact, positive or negative, on short-term cognitive functioning when compared to alendronate.

[Plasma concentrations of folic acid, vitamin B12 and progesterone of cyclic bitches, bitches during pregnancy and induced abortion and bitches with pyometra]. / Plasmakonzentrationen von Folsäure, Vitamin B12 und Progesteron während des Sexualzyklus, Gravidität, Abortinduktion und Pyometra bei der Hündin.

The aim of this study was to investigate the plasma concentrations of folic acid, vitamin B12 and progesterone at different stages of the sexual cycle and pregnancy, during induced abortion and in bitches with pyometra. Bitches (n = 97) were assigned to groups as follows: a) oestrous cycle (n = 42) b) pregnancy (n = 25) c) induction of abortion (n = 10 and d) pyometra (n = 20). Oestrous cycle stages were determined by vaginal inspection and cytology. Pregnancies were estimated by ultrasound (5.0 Mhz; linear transducer; Schimadzu) at days 15-25, 35-45 and 46-63 of pregnancy. Treatments for the induction of abortion were started between days 25 and 35 after mating (5 microg/kg cabergoline daily, Galastop; 5-10 microg/kg Alfaprostol every other day, Gabbrostim). Diagnosis of pyometra was confirmed by ultrasound and vaginoscopy. Folic acid and vitamin B12 concentrations did not differ among different stages of the oestrous cycle. The mean concentration of folic acid during early pregnancy (days 15-25) exceeded levels of later stages (days 46-63): 9.4 +/- 3.7 microg/ml and 4.7 +/- 1.8 microg/ml, respectively (p < 0.01). A positive correlation between folic acid and vitamin B12 was determined in pregnant dogs ( r = 0.925; p < 0.02). Before the induction of abortion, the concentration of folic acid was 9.6 +/- 5.2 microg/ml; during abortion it decreased to 5.0 +/- 3.2 microg/ml (p < 0.01). A significant correlation (r = 0.925; p < 0.02) between progesterone and folic acid was obtained in bitches with abortion. The mean concentration of folic acid in bitches with pyometra significantly differed from that of bitches at different stages of the oestrous cycle (p < 0.05). The mean concentration of folic acid was significantly lower in metoestrous bitches when compared to bitches with pyometra (p < 0.05). The decrease of serum concentrations of folic acid during pregnancy and induced abortion show that fetal growth and abortion caused higher consumption of folic acid. Concerning bitches did not show any deficiency symptoms, which is why it can be concluded that this decrease is physiological.

Matrix metalloproteinase 2 (MMP-2) and tissue transglutaminase (TG 2) are expressed in periglandular fibrosis in horse mares with endometrosis.

Periglandular arrangement of myofibroblasts, associated with the deposition of extracellular matrix (ECM), is a cardinal feature of endometrosis in mares. We hypothesized that a disturbance in the expression of matrix degrading enzymes such as matrix metalloproteinases (MMP's) and matrix cross-linking proteins might lead to an imbalance in deposition and degradation of extracellular matrix components and thereby accentuate degeneration. Therefore, distributions of MMP-2, capable of collagen IV and laminin degradation, and tissue transglutaminase (TG2), a cross-linker of extracellular matrix proteins, were investigated by means of immunohistochemistry on uterine biopsies of healthy mares and animals with endometrosis. It was illustrated that both proteins were present in fibrotic regions of affected endometria, and that they were in most cases colocalized. Periglandular MMP-2 expression was significantly associated with dilated and fibrotic uterine glands. Furthermore, MMP-2 and TG 2 were demonstrated in the stratum compactum of healthy and endometrotic endometria. Gelatin zymography proved that active and inactive pro-form of MMP-2 were present in all examined samples with significantly higher amounts of total and active MMP-2 in affected endometria. TG 2-activity, determined by an in situ assay, was found in cases of severe periglandular fibrosis. We suggest that both enzymes play a major role in changes that occur in ECM homeostasis in endometrial fibrotic regions.

Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration.

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.

[Suitability of sonographic evaluation of ovarian dynamics and uterine involution for prediction of postpartum fertility in the cow]. / Eignung der sonographischen Beurteilung von Ovardynamik und Uterusinvolution zur Fertilitätsprognose im Puerperium des Rindes.

To investigate the suitability of sonography for prediction of puerperal fertility, we used 34 cows with normal puerperium. For this purpose, the animals were examined gynecologically and by transrectal sonography at 10-day intervals between day 15 and day 45 p.p. as well as at oestrus, when they were inseminated. We judged uterine involution by diameter and sonographic structure of the uterine cross-section and ovarian activity by the presence of follicles and corpora lutea. Cows, which became pregnant after first insemination p.p., were assigned to group G1 (n = 16) and animals, which failed to conceive, to group G2 (n = 18). The cows of G1 obtained significantly better results concerning both uterine involution and ovarian activity than cows of G2. Thus, diameter of the uterine horn decreased faster and sonographic structure of the uterine cross section returned markedly earlier to the nonpregnant appearance in G1 than in G2. Group G1 also showed a significantly higher percentage of dominant follicles at all examinations. The results of the study show a distinct relationship between uterine involution and ovarian activity and puerperal fertility. But it has also to be resumed that the prediction of fertility for individuals will always be restricted by other reasons causing failure of conception.

False-positive urine beta-HCG in a woman with a tubo-ovarian abscess.

Modern urine beta-human chorionic gonadotropin (HCG) assays that use enzyme-linked immunosorbent assay (ELISA) technology are sensitive and specific for diagnosing pregnancy, both intrauterine and ectopic, and have become indispensable to the practice of Emergency Medicine. A urine HCG test is often relied on by the Emergency Physician as a critical component in the diagnostic regimen of a patient with a possible ectopic pregnancy. We report a case of a false-positive urine beta-HCG test in a patient with a ruptured tubo-ovarian abscess. Though false-positive pregnancy tests with tubo-ovarian abscesses have previously been reported with older methods of HCG detection, we believe that this is the first case where the pregnancy test was the modern ELISA type. The mechanism for the false-positive reaction in this case is unknown, but time may show that the ELISA test kit, like its predecessors, may occasionally give a false-positive reaction in this class of patients.

Transcriptional regulation by changes in tonicity.

Most organisms respond to a hypertonic environment by accumulating small organic solutes. In contrast to high concentrations of electrolytes, the small organic solutes do not perturb the activity of enzymes and other macromolecules within the cell. When the renal medulla becomes hypertonic during antidiuresis, multiple signaling pathways are activated. Here, we review the role of tonicity responsive enhancers (TonE) binding protein (TonEBP), a transcription factor activated in hypertonic cells. The activation of TonEBP by hypertonicity results from its translocation to the nucleus as well as an increase in TonEBP mRNA and protein. TonEBP may have a role beyond the response to tonicity since it is highly expressed in activated lymphocytes and in developing tissues.

Lectin binding patterns of uterine glands in mares with chronic endometrial degeneration.

OBJECTIVE: To evaluate changes of glycoconjugate in uterine glands of endometrial tissues obtained from mares. ANIMALS: adult mares. PROCEDURE: Uterine biopsy samples were collected during the breeding season and analyzed histologically for signs of chronic endometrial degeneration. Stage of the estrous cycle was established, using clinical examination and determination of hormonal status. Uterine tissue samples were analyzed, using lectin histochemical and immunohistochemical techniques (estrogen and progesterone receptors). Connective tissues were stained to determine alterations of ground substance in periglandular fibrosis. RESULTS: Of 50 mares, 30 (60%) were classified as normal or having modest alterations, and 20 (40%) were classified as having moderate or severe endometrial degeneration. In normal equine endometrium, several lectins (Helix pomatia agglutinin, Lotus tetragonolobus agglutinin, Ricinus communis I agglutinin, Ulex europaeus agglutinin, and wheat germ agglutinin) bound to glycoconjugates of the luminal epithelium and openings of uterine glands. Lectin binding patterns of cystic dilated glands or fibrotic glands in endometrial samples were remarkably strong, whereas normal surrounding cells remained unstained. Lotus tetragonolobus lectin was not suitable for detecting endometrial alterations. Connective tissues stained with Alcian blue and results of Hale colloidal-iron binding revealed acidic ground substance in periglandular fibrosis. Estrogen and progesterone receptors were evenly distributed in healthy and affected endometrial samples. CONCLUSIONS AND CLINICAL RELEVANCE: Glycoconjugate patterns of uterine glands were altered in mares with chronic endometrial degeneration. Therefore, uterine secretions are likely to be altered. These changes are not induced by changes in content of estrogen and progesterone receptors in endometrial tissues.

Association of endometriosis in horses with differentiation of periglandular myofibroblasts and changes of extracellular matrix proteins.

Periglandular fibrosis and cystic dilation of uterine glands are associated with equine endometriosis. The presence of extracellular matrix proteins (collagen type I, III and IV, laminin and fibronectin) in healthy and endometriotic specimens was demonstrated by immunohistochemistry. The distribution of collagen I, but not collagen III, was dependent on the stage of the oestrous cycle. The arrangement of collagen I and collagen III in endometriotic specimens was similar to that in normal endometrium. In periglandular fibrosis, collagen IV, laminin and fibronectin deposition outside the basement membrane was observed. In these regions, stromal cells were characterized immunohistochemically as myofibroblasts because of their expression of a-smooth muscle actin, and occasionally tropomyosin and desmin. Periglandular differentiation of contractile cells could be interpreted as a reaction to support the extrusion of secretions in cystic dilated glands. Moreover, the changes of extracellular matrix proteins are characteristic for neoplastic lesions, although further development of endometriosis to benign or malignant tumours is not known in horses. Knowledge of the factors responsible for these fibroblastic modulations may be the key to explaining the pathogenesis of endometriosis.