[Diagnostic utility of the anti-Mullerian hormone in companion animals]. / Diagnostischer Nutzen des Anti-Müller-Hormons in der Kleintierpraxis.

The anti-Mullerian hormone is a glycoprotein secreted by Sertoli cells in males and granulosa cells in females. The initial identification of this hormone in canine and feline serum was achieved in 2011. Meanwhile, a variety of studies have demonstrated its clinical significance as a tool in the endocrine diagnosis. This review summarizes the current knowledge about anti-Mullerian hormone in small animal reproduction and describes future opportunities for its diagnostic usage.

Anti-Müllerian hormone concentrations in female rabbits and its relation to spay status, pseudopregnancy and ovarian follicle numbers.

Anti-Müllerian hormone (AMH), known for its role during foetal sexual differentiation, is secreted by the Sertoli cells in males and the granulosa cells in females during post-natal life. As serum AMH concentrations correlate with follicle numbers, AMH is utilized as a marker of ovarian reserve in many species. In dogs and cats, AMH is used as a diagnostic tool to determine spay or neuter status. In the available literature, no research regarding serum AMH levels in rabbits has been published yet. The objectives of the present study were to (1) measure serum AMH concentrations in female rabbits and investigate the value of AMH as a diagnostic tool to differentiate between spayed and intact does and (2) relate measured AMH levels to pseudopregnancy and ovarian follicle numbers. For AMH measurement, serum samples were obtained from sexually intact (n = 64) and spayed (n = 22) female rabbits. Spayed does were of various breeds; intact rabbits were Zika hybrid rabbits. In the intact does, AMH measurement was complemented by determination of progesterone levels, gynaecological examination and histopathological evaluation of the uterus and ovaries, including follicle counts. Serum AMH and progesterone concentrations were measured using a human-based chemiluminescence immunoassay (CLIA) and an enzyme-linked fluorescence assay (ELFA), respectively. Depending on progesterone levels, sexually intact does were classified into follicular (n = 52) or luteal phase (n = 12). Median serum AMH levels were 1.53 ng/ml (range 0.77-3.36 ng/ml) in intact and 0.06 ng/ml (range ≤0.01-0.23 ng/ml) in spayed does. AMH concentrations between the intact and spayed rabbits differed significantly and did not overlap (p < .001). Receiver operating characteristic (ROC) curve analysis yielded a sensitivity and specificity of 100% for a cut-off level of 0.50 ng/ml. Follicular or luteal phase had no significant influence on measured AMH levels (t = 0.061, df = 62, p = .951). While the number of secondary follicles correlated significantly with AMH concentrations (rs  = 0.410, p = .001), the number of primary or antral follicles did not (rs  = 0.241, p = .055 and rs  = 0.137, p = .281, respectively). In conclusion, a single determination of serum AMH concentrations was adequate to distinguish spayed from intact female rabbits. Among sexually intact individuals, whether does were in follicular or luteal phase had no significant influence on measured serum AMH concentrations. The relationship between small growing follicles and AMH levels as described in other species could be partially confirmed, as secondary follicles correlated significantly with AMH.

Anti-Müllerian hormone concentrations in queens throughout the estrous cycle.

Studies in cats and dogs have proven the usefulness of anti-Müllerian hormone (AMH) as a diagnostic tool to determine the castration status or to diagnose ovarian remnant syndrome. Yet the secretion pattern of AMH over the estrous cycle in queens has not been investigated so far. Seven healthy sexually intact female cats were examined daily for signs of estrous behavior over a trial period of 4 months. Five queens showed regular estrous behavior, 1 queen was mated in her first heat and 1 queen never showed any signs of heat. To distinguish between inter-estrus and metestrus progesterone levels were determined. Serum samples for AMH and progesterone measurement were collected from the regular cycling queens in late anestrus, at several times during heat, inter-estrus and metestrus, from the mated queen during her first heat and during pregnancy, and in the acycling queen at various times during the trial period. The measured AMH values in anestrus were significantly (P < 0.05) higher than in heat (P < 0.001), metestrus (P = 0.12) and inter-estrus (P = 0.449). In anestrus the median AMH levels were 10.26 ng/ml (range 4.96 to 22.90 ng/ml), in heat 5.97 ng/ml (range 3.32- 22.96 ng/ml), in inter-estrus 10.47 (range 3.35-22.96 ng/ml) and in metestrus 6.38 ng/ml (range 4.50-10.75 ng/ml. The pregnant cat showed median AMH concentrations of 6.47 ng/ml (range 5.60-9.80 ng/ml) during her pregnancy. The acycling queen had solely low AMH values with a median concentration of 0.39 ng/ml. In conclusion there were high variations of the AMH levels among and within the individual cats and between heat cycles in the single cat. Remarkable high AMH concentrations were measured in the younger queens of the study in their first estrous cycles and also in anestrus, when less ovarian activity is expected. Further studies are necessary to emphasize the reasons for these high AMH concentrations especially in young queens.

Anti-Müllerian hormone as a diagnostic tool to identify queens with ovarian remnant syndrome.

OBJECTIVES: Ovarian remnant syndrome (ORS) is suspected when heat signs occur in spayed individuals, but further diagnostic procedures are necessary to exclude other possible oestrogen sources, such as the adrenal gland or exogenous supplementation. Anti-Müllerian hormone (AMH), secreted by granulosa cells or Sertoli cells, serves to differentiate sexually intact from gonadectomised animals and has been described in dogs as a tool for diagnosing ORS. The aim of this study was to evaluate if AMH determination can be used to diagnose ORS in cats. METHODS: AMH was measured with a chemiluminescence immunoassay in serum samples of 15 sexually intact, 9 spayed and 16 cats with a history of heat signs after spaying. Abdominal ultrasound (n = 13), vaginal smears (n = 7), progesterone measurement (n = 5) and laparotomy (n = 14) were used to determine the presence of ovarian tissue. After surgery, a histological examination of the obtained tissue was performed in the cats with suspected ORS. RESULTS: In 15 cats with ORS the AMH serum concentrations were significantly higher than in spayed cats (n = 10; P = 0.025) and significantly lower than in sexually intact cats (n = 15; P = 0.001). Among the cats with ORS, the highest AMH serum concentrations were measured in the queens with cystic ovarian alterations and in one cat from which a whole ovary was obtained. The cat with the lowest AMH serum concentration had a simultaneous high progesterone serum concentration. Cats with ORS did not show any heat signs after surgical removal of the ovarian tissue. CONCLUSIONS AND RELEVANCE: A single determination of AMH in blood serum is a useful diagnostic tool for the diagnosis of ORS in cats, regardless of the hormonal activity of the remnant ovarian tissue.

Anti-Müllerian hormone, testosterone, and insulin-like peptide 3 as biomarkers of Sertoli and Leydig cell function during deslorelin-induced testicular downregulation in the dog.

The role of anti-Müllerian hormone (AMH) and insulin-like peptide 3 (INSL3) in male infertility is not fully understood. We used the downregulated testis as a model of gonadotropin-dependent infertility. Serum testosterone and AMH concentrations were studied in five adult male Beagles implanted (day 0) with 4.7 mg deslorelin (Suprelorin®, Virbac) (DES group). Testicular expression of LH receptor (LHR) and androgen receptor (AR), AMH, type 2 AMH receptor (AMHR2), INSL3 and its receptor (RXFP2) was evaluated 112 days (16 weeks) after deslorelin treatment by qPCR and immunohistochemistry, and compared to untreated adult (CON, n = 6) and prepubertal (PRE, n = 8) dogs. Serum testosterone concentration decreased significantly by the onset of aspermia on study day 14 (four dogs) or day 21 (one dog), and was baseline on day 105 (week 15). In contrast, serum AMH started to increase only after the onset of aspermia and reached the maximum detectable concentration of the assay by day 49-105 in individual dogs. Testicular LHR gene expression in DES was lower than in CON and PRE (P < 0.0001), while AR gene expression in DES was similar to CON and significantly higher than PRE (P < 0.0001). Testicular AMH expression in DES was intermediate compared to the lowest mRNA levels found in CON and the highest in PRE (P ≤ 0.006). AMHR2 gene expression was similar between groups. AMH protein was detected in Sertoli cells only, while AMHR2 immunoreactivity was principally detected in Leydig cells which appeared to be increased in DES. INSL3 and RXFP2 gene expression was significantly downregulated in the DES testis along with noticeably weak Leydig cell immunosignals compared to CON. In conclusion, deslorelin treatment caused testicular LH insensitivity without affecting androgen sensitivity, and de-differentiation of Sertoli and Leydig cells. In DES, upregulation of the AMH-AMHR2 feed-back loop and downregulation of the INSL3-RXFP2 feed-forward loop are paracrine-autocrine mechanisms that may additionally regulate testosterone production independent of gonadotropins. Our results support AMH and INSL3 as unique biomarkers and paracrine-autocrine regulators of testis function involved in the intimate interplay between Sertoli and Leydig cells.

[Birth of a vital puppy following prolonged whelping in a Labrador Retriever]. / Geburt eines lebenden Labradorwelpen nach deutlich verlängerter Geburt.

An 8-year-old primiparous Labrador Retriever bitch was presented to the clinic 46 hours following the delivery of 2 vital and one stillborn puppy due to an increase of vaginal discharge. At the veterinary clinic, the dog spontaneously delivered an additional healthy puppy without any obstetric support. The findings of the general and gynaecological examinations as well as results of blood analyses were within normal limits. This case shows that extremely prolonged whelping process may be a rare but possible phenomenon in bitches. However, it is recommended to re-evaluate dam and fetuses regularly in case the inter-whelping interval exceeds the normal time span in order to recognize further signs of dystocia and induce obstetric support when clinically indicated.

Anti-Müllerian hormone in dogs and cats reproduction.

The anti-Müllerian hormone (AMH) is a glycoprotein secreted by Sertoli cells in males and granulosa cells in females. It has first been determined in blood serum of dogs and cats by Place et al. in 2011 with the use of a human-based ELISA test. Meanwhile, different immunoassays have been validated for AMH determination in animals and a variety of studies have demonstrated the clinical significance of AMH. This review summarizes the current knowledge about AMH in dogs and cats and describes future opportunities for its diagnostic use.

Semen parameters and testicular dimensions in small breed dogs below ten-kilogram bodyweight.

Because of few available data on semen parameters in small breed dogs, the study aimed to analyse semen and measure testes of dogs ≤10.0 kg bodyweight. Semen was collected from 41 fertile stud dogs, which were divided based on bodyweight: group 1 ≤ 5.0 kg and group 2 between 5.1 and 10.0 kg. Median values for ejaculate volume (group 1: x ~ 1.2 ml; group 2: x ~ 2.2 ml), total sperm output (group 1: x ~ 110.7 × 106 ; group 2: x ~ 215.1 × 106 ) and testicular volume (group 1: left testicle x ~ 2.8 ml, right testicle x ~ 2.7 ml; group 2: left testicle x ~ 5.5 ml, right testicle x ~ 5.0 ml) were lower in group 1 compared to dogs of group 2 (p = .001; p = .001; both testes: p < .001). There was no difference in sperm concentration (p = .985). Based on these results, introduction of an additional weight group to the commonly used reference values is recommended, since values for ejaculate volume, total sperm output and testicular dimensions for dogs ≤5.0 kg bodyweight differed significantly from values of dogs with a bodyweight from 5.1 to 10.0 kg.

Changes in anti-Müllerian hormone concentrations in bitches throughout the oestrous cycle.

Studies in female dogs proved the measurement of anti-Müllerian hormone (AMH) as a diagnostic tool to distinguish ovarioectomized from intact females or to diagnose ovarian remnant syndrome. Furthermore, its usability to diagnose a granulosa cell tumour and predict litter size was also investigated in female dogs. Although serum AMH was previously shown to increase during the transition from anoestrus to pro-oestrus in dogs, changes in AMH concentration over the entire oestrous cycle have not yet been determined. The aim of this study was to investigate the secretion pattern of AMH throughout the oestrous cycle in non-pregnant bitches. As a preliminary step, we tested our assay by measuring AMH concentrations in serum samples of 19 intact bitches (10 in anoestrus, 3 in pro-oestrus, 3 in oestrus, 3 in metoestrus) and 19 spayed females of different age and breed. For the main study, 20 healthy female dogs were examined (10 Beagles, and 10 Labrador crossbreeds, body weight 10-28 kg, age 1-6 years) during a normal oestrous cycle. Serum samples were collected in late anoestrus, at several times during pro-oestrus and oestrus, as well as 28 days and 4.5 months after ovulation. Blood collection was combined with a gynaecological examination including progesterone measurement. Serum concentration of AMH was determined using a chemiluminescence immunoassay validated for dog serum. In the preliminary test, intact bitches in various stages of the oestrous cycle had significantly higher serum AMH levels (0.19-1.45 ng/ml) than spayed females (0.01-0.06 ng/ml, P < 0.001). In cycling Beagle and Labrador crossbreed bitches participating in our main study, AMH concentrations were between 0.09 and 2.65 ng/ml with higher AMH concentrations in Beagles but overall high inter-individual and intra-individual variation. The highest AMH values were reached in the last three weeks before the onset of heat until six days before ovulation, and the lowest AMH concentrations were recorded during preovulatory oestrus in 6, metoestrus in 7 or anoestrus in 7 dogs. Serum AMH concentrations increased significantly from late anoestrus up to six days before ovulation and decreased significantly over the last three days before ovulation. A further significant decrease occurred from the last days of oestrus to metoestrus and mid anoestrus. This study shows that the used AMH assay can clearly distinguish between intact and spayed females and that the serum AMH pattern over the oestrous cycle is similar in all bitches, but with high variation among the breeds and among and within bitches. These changes in the AMH concentration are a challenge in timing serum sample collection and interpreting AMH values in bitches. Further studies are necessary to emphasize the factors influencing the AMH concentration.

Strategic and Statistical Considerations on the QT Assessment of Volasertib.

Volasertib is a selective cell cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Polo-like kinase (Plk). A potential for prolonged QT intervals was indicated with volasertib in preclinical studies and preliminary clinical data. As a result, electrocardiograms (ECGs) have been collected in all volasertib clinical trials to monitor potential cardiac effects. This article describes strategic and statistical methods prospectively planned to perform an integrated analysis of ECG data from available trials to evaluate volasertib's effect on cardiac repolarization, as reflected by changes in the duration of QT interval and other ECG-related endpoints. Methods to effectively cope with heterogeneity between trials (ie, differences in study designs) are discussed. These strategies may be useful for other investigational drugs for which QT risk assessment is required, but a thorough QT/QTc trial is not feasible, resulting in the need for an alternative approach. Volasertib therapy relevantly prolonged adjusted mean QTcF change from administration baseline following the first and subsequent infusions. The integrated analysis revealed that the volasertib effects on the mean QTc changes from baseline were transient and had resolved at 24 hours after start of the first infusion. There was no evidence for a long-term impact on the QTcF interval following multiple infusions with volasertib.

The sodium glucose cotransporter 2 inhibitor empagliflozin does not prolong QT interval in a thorough QT (TQT) study.

BACKGROUND: Empagliflozin is a potent, selective sodium glucose cotransporter 2 (SGLT2) inhibitor in development as an oral antidiabetic treatment. This QT interval study assessed potential effects of empagliflozin on ventricular repolarisation and other electrocardiogram (ECG) parameters. METHODS: A randomised, placebo-controlled, single-dose, double-blind, five-period crossover study incorporating a novel double-placebo period design to reduce sample size, while maintaining full statistical power. TREATMENTS: single empagliflozin doses of 25 mg (therapeutic) and 200 mg (supratherapeutic), matching placebo and open-label moxifloxacin 400 mg (positive control). Triplicate 12-lead ECGs of 10 second duration were recorded at baseline and during the first 24 hours after dosing. The primary endpoint was mean change from baseline (MCfB) in the population heart rate-corrected QT interval (QTcN) between 1-4 hours after dosing. RESULTS: Thirty volunteers (16 male, 14 female, mean [range] age: 34.5 [18-52] years) were randomised. The placebo-corrected MCfB in QTcN 1-4 hours after dosing was 0.6 (90% CI: -0.7, 1.9) ms and -0.2 (-1.4, 0.9) ms for empagliflozin 25 mg and 200 mg, respectively, below the ICH E14 defined threshold of regulatory concern 10 ms. Assay sensitivity was confirmed by a placebo-corrected MCfB in QTcN 2-4 hours post-dose of 12.4 (10.7, 14.1) ms with moxifloxacin 400 mg. Empagliflozin tolerability was good for all volunteers; 23.3% experienced adverse events (AEs) with empagliflozin and 27.6% with placebo. The most frequent AE was nasopharyngitis. CONCLUSIONS/INTERPRETATION: Single doses of empagliflozin 25 mg and 200 mg were not associated with QTcN prolongation and were well tolerated in healthy volunteers. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01195675.

The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon ß (IFNß) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNß promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNß gene.

Levetiracetam: safety and efficacy in neonatal seizures.

PURPOSE: Neonatal seizures are common, especially in prematurity. Phenobarbital (PB) currently represents the antiepileptic drug (AED) of choice, despite being related to increased neuronal apoptosis in animal models and cognitive impairment in human subjects. Levetiracetam (LEV) may have a more favorable profile since it does not cause neuronal apoptosis in infant rodents. METHODS: In a prospective feasibility study, LEV was applied as first-line treatment in 38 newborns with EEG-confirmed seizures, after ruling out hypoglycemia, hypocalcaemia, hypomagnesaemia and pyridoxin dependency. Initial intravenous doses of 10 mg/kg LEV were gradually increased to 30 mg/kg over 3 days with a further titration to 45-60 mg/kg at the end of the week. Acute intervention with up to 2 intravenous doses of PB 20 mg/kg was tolerated during LEV titration. LEV was switched to oral as soon as the infants' condition allowed. Based on clinical observation, EEG tracings (aEEG/routine EEGs), and lab data, drug safety and anticonvulsant efficacy were assessed over 12 months. RESULTS: In 19 newborns a single PB dose of 20 mg/kg was administered, while 3 newborns received 2 PB doses. 30 infants were seizure free under LEV at the end of the first week and 27 remained seizure free at four weeks, while EEGs markedly improved in 24 patients at 4 weeks. In 19 cases, LEV was discontinued after 2-4 weeks, while 7 infants received LEV up to 3 months. No severe adverse effects were observed. CONCLUSIONS: These results illustrate the safety of LEV treatment in neonatal seizures, including prematurity and suggest LEV anticonvulsant efficacy. Additional PB treatment admittedly constitutes a methodological shortcoming due to the prolonged anticonvulsive efficacy of PB. Double blind prospective controlled studies and long-term evaluation of cognitive outcome are called for.