Using Vaginal Impedance Measurement to Identify Proestrus in Rats Given Luteinizing Hormone Releasing Hormone (LHRH) Agonist.

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

Effect of Punica granatum, Citrus limon and their combinations on the plasma Gonadotropins in female rabbits.

Fruits produce revitalizing effects, hence the impact of Punica granatum, Citrus limon and their combinations have been investigated on the plasma levels of gonadotropin, testosterone and sexual development capacity in female rabbits. Ninety female rabbits were randomly assigned into nine groups, each comprising of ten animals. One group was given saline and designated as control. Three groups were given P. granatum 2mL /kg, 5mL/kg, 8mL/kg, other three groups received C. limon 0.2mL/kg, 0.4mL/kg, 0.6mL/kg respectively, remaining groups received C. limon and P. granatum in combination i.e. 0.4mL/kg C. limon + 5mL/kg P. granatum and 0.2mL/kg C. limon + 8mL/kg P. granatum. Juices were administered once daily by mouth from day 0 of pups delivered to postnatal day15. Blood samples were gathered from ear vein at day11 and day15. There was significant increase in follicle stimulating hormone by P. granatum at 5 and 8mL/kg on day 11 and 15, by C. limon at 0.4 and 0.6mL/kg on day11, 0.4mL/kg at day15, by combination doses of C. limon and P. granatum 0.4 +5mL/kg at day 11, 0.4+5 mL/kg and 0.2 + 8mL/kg at day15. There was also significant increase in luteinizing hormone by P. granatum at 2, 5 and 8mL/kg and by C. limon 0.4mL/kg at day11. There was highly significant increase on day 11 in LH at combination doses of C. limon and P. granatum 0.4 + 5mL/kg. There was significant increase in testosterone level by P. granatum at 2, 5 and 8mL/kg on day 11 and 5mL/kg on day15 and highly significant increase at 2 and 8mL/kg. C. limon caused significant increase in testosterone at 0.4 mL/kg on day11, 0.2 and 0.6mL/kg on day 15 and highly significant increase at 0.4mL/kg on day15. Whereas combinations doses of C. limon and P. granatum at 0.4+5mL/kg caused highly significant increase in testosterone level as compare to control. Results of present study revealed increase in plasma gonadotropin and testosterone levels showing increase in sexual capacity of female rabbits which could be mainly accounted for high vitamin C and flavonoids contents of these juices.

The need for more aggressive therapy for men with Gleason 9-10 disease compared to Gleason ≤8 high-risk prostate cancer.

PURPOSE: To evaluate the outcomes of prostate cancer patients with high-risk disease stratified by Gleason Score (GS) (GS ≤8 vs GS ≥9) treated with external beam radiotherapy (EBRT). METHODS: The medical records of patients who underwent EBRT between 2003 and 2011 and had nonmetastatic high-risk disease were analyzed retrospectively. Patients were treated with EBRT and all patients received a dose ≥7,560 cGy. Androgen deprivation therapy was given in most patients (90%). RESULTS: A total of 155 patients were identified (GS ≤8 n = 104, GS ≥9 n = 51), and they had a median presenting prostate-specific antigen (PSA) of 14.7 ng/mL. At a median follow-up of 69 months, the 7-year biochemical failure-free survival was 59.1% in those with GS ≥9 and 69.2% in those with GS ≤8 (p = 0.12). On MVA, Gleason 9-10 (HR 1.83, p = 0.08) was not associated with an increased risk of biochemical recurrence, while a PSA >20 ng/mL (HR 2.39, p = 0.04) was associated with an increased likelihood of biochemical recurrence. Patients with GS ≥9 were noted to have worse 7-year distant metastatic-free survival (79.6% vs 90.5% p = 0.02) and cancer-specific survival (88.5% vs 97.9%, p = 0.006). On MVA, GS ≥9 was a significant indicator of distant metastatic failure and cancer-related death. Seven-year overall survival rates remained similar between the groups. CONCLUSIONS: In this high-risk cohort, patients with GS 9-10 had significantly worse prostate cancer-related outcomes than other high-risk patients, suggesting that this group may warrant more aggressive treatment modalities than their high-risk counterparts.

Is Gleason Grade 5 Prostate Cancer Resistant to Conventional Androgen Deprivation Therapy?

Synthesis of data from randomized trials suggests that long-term versus short-term androgen deprivation therapy may be most effective in men with Gleason grade 4 prostate cancer (PCa) and less or ineffective in men with Gleason grade 5 PCa.

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17ß-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Risks, benefits, and approaches to hormonal blockade in prostate cancer. Highlights from the European Association of Urology Meeting, March 20-24, 2015, Madrid, Spain.

Several abstracts presented at the 2015 European Association of Urology Meeting highlighted new developments in hormone therapy for prostate cancer management. One abstract described how the luteinizing hormone-releasing hormone (LHRH)/gonadotropin-releasing hormone (GnRH) agonist leuprolide, but not the LHRH/GnRH antagonist degarelix, induced plaque instability in a mouse model. A second abstract showed that in patients with a history of severe cardiovascular disease, degarelix was associated with fewer cardiovascular events than treatment with an LHRH agonist. A third abstract showed how primary androgen-deprivation therapy was linked with increased all-cause mortality in a US registry. A fourth abstract showed that in the ANAMEN study, cognitive performance was not significantly affected by 6 months of treatment with GnRH agonists. Last, a fifth abstract showed that low-dose prednisone, with or without abiraterone, was associated with an overall low incidence of corticosteroid-associated adverse events.

Endurance exercise attenuates cardiotoxicity induced by androgen deprivation and doxorubicin.

Doxorubicin (DOX) is associated with cardiac dysfunction and irreversible testicular damage. Androgen deprivation therapy (ADT) is administered prior to DOX treatment to preserve testicular function. However, ADT may exacerbate DOX-induced cardiac dysfunction. Exercise is cardioprotective, but the effects of exercise on cardiac function during combined ADT and DOX treatment are currently unknown. In this study, male Sprague-Dawley rats were randomly assigned to experimental groups: control (CON), ADT, DOX, or ADT+DOX. Animals received ADT or control implants on days 1 and 29 of the 56-day protocol. Animals remained sedentary (SED) or engaged in treadmill endurance exercise (TM) beginning on day 1. On day 15, the animals received DOX at 1 mg·(kg body mass)(-1)·d(-1) by intraperitoneal injection for 10 consecutive days, or an equivalent volume of saline. On day 57, cardiac function was assessed in vivo and ex vivo. Animals treated with DOX alone, or with combined ADT+DOX, showed significant (P < 0.05) reductions in left ventricular developed pressure (-21% and -27%), maximal rate of pressure development (-29% and -32%), and maximal rate of pressure decline (25% and 31%), respectively when compared with the sedentary control animals. Endurance exercise training attenuated (P > 0.05) cardiac dysfunction associated with combined ADT+DOX treatment, indicating that exercise during simultaneous ADT+DOX treatment is cardioprotective.

Long-term outcome for prostate cancer using pseudo pulse-dosed rate brachytherapy, external beam radiotherapy, and hormones.

PURPOSE: We report the long-term outcomes of pulse-dose rate (PDR) brachytherapy used in a nonstandard style (pseudo-PDR) with an high-dose rate brachytherapy technique in conjunction with external beam radiotherapy (EBRT) and hormonal manipulation on prostate cancer (PC). METHODS AND MATERIALS: We treated 253 patients with Stage T1-T3 N0M0 PC, between December 1999 and March 2006. All patients received neoadjuvant androgen deprivation for a median 6 months. Treatment consisted of three pulses of pseudo-PDR brachytherapy to a median dose of 18Gy with 50.4Gy in 28 fractions of EBRT. RESULTS: At a median 6 years followup, (range, 1-11 years), 5-year overall survival was 92%, and PC-specific survival was 96%. The 5-year biochemical control (biochemical no evidence of disease) by the Phoenix definition for low-, intermediate-, and high-risk groups was 95%, 90%, and 71%, respectively (p<0.00001). At 6 years, the incidence of Radiotherapy Oncology Group Grade 2 and 3 genitourinary toxicity was 1% and 6%; Radiotherapy Oncology Group Grade 2 and 3 gastrointestinal toxicity was 4% and 0%. Erectile preservation at 3 years was 58%. The Phoenix definition best predicted clinical failure with a high specificity (94%). CONCLUSIONS: Pseudo-PDR brachytherapy plus EBRT with limited neoadjuvant hormonal manipulation is an effective treatment option in localized PC, with minimal and tolerable morbidity and provides excellent control. This technique of a modified PDR-delivery technique appears as effective as high-dose rate therapy.

First evidence of ovulation induced by oral LH agonists in healthy female volunteers of reproductive age.

CONTEXT: Two new low-molecular-weight LH agonists (Org 43553 and Org 43902) were shown to induce ovulation in preclinical experiments. OBJECTIVE: Our objective was to assess the safety, pharmacokinetics, and pharmacodynamics of Org 43553 and Org 43902 when administered to healthy females. DESIGN AND SETTING: Org 43553 and 43902 studies were randomized, placebo-controlled, single-rising-dose first-in-human trials, which included 159 healthy female volunteers. Part 1 of the studies assessed the safety and pharmacokinetics. Part 2 evaluated the pharmacodynamics effect of a single oral dose of Org 43553 (25-900 mg) or Org 43902 (30-300 mg) to induce ovulation after the development of a large preovulatory follicle, whereas the endogenous LH surge was suppressed due to GnRH antagonist treatment while follicular development was supported with recombinant FSH. RESULTS: Org 43553 and 43902 were safe and well tolerated. Both compounds showed a fast absorption after oral intake, with peak concentrations reached within 0.5 to 1 hour. The elimination half-life of Org 43553 was 30 to 47 hours and that of Org 43902 was 17 to 22 hours. Ovulation induction confirmed by midluteal progesterone rise ≥15 nmol/L was proven in both studies, also when excluding subjects with an endogenous LH rise. The minimal effective dose for ovulation induction was 300 mg in both studies and resulted in an ovulation rate of 83% and 82%, respectively. CONCLUSIONS: These first proof-of-concept studies both demonstrated that a single oral intake of an low-molecular-weight LH agonist induces ovulation of the preovulatory follicle in pituitary-suppressed female volunteers of reproductive age.

Effects of genistein on gonadotropic cells in immature female rats.

The effects of genistein on pituitary gonadotropic cells of immature female rats were examined and compared to actions of the synthetic estrogen, 17α-ethynylestradiol. Immature female rats received 50mg/kg/bw of genistein in dimethylsulfoxide (DMSO) subcutaneously (s.c.) daily for 3 days at 18, 19 and 20 days of age. A second group was injected with 1µg/kg of 17α-ethynylestradiol in olive oil in the same schedule. The genistein control group received DMSO only, while 17α-ethynylestradiol controls were given sterile olive oil only. Changes in cell number per mm(2), cell volume and volume density of follicle-stimulating (FSH) and luteinizing (LH) immunolabeled cells were evaluated by morphometry and stereology. Genistein induced significant increases in the number of FSH cells (by 21%) and LH cells (by 20%) per mm(2) compared to corresponding controls. Volumes of FSH and LH cells were significantly increased by 19.7% and 20% and their volume densities by 20% and 20.2%, respectively. Estradiol markedly affected gonadotropes in the same manner, but to a greater extent. It can be concluded that genistein acted as an estrogenic agonist in the pituitaries of immature female rats, and as such, stimulated gonadotropic cells.

Effect of priming injections of luteinizing hormone-releasing hormone on spermiation and ovulation in Gϋnther's toadlet, Pseudophryne guentheri.

BACKGROUND: In the majority of vertebrates, gametogenesis and gamete-release depend on the pulsatile secretion of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus. Studies attempting to artificially stimulate ovulation and spermiation may benefit from mimicking the naturally episodic secretion of LHRH by administering priming injections of a synthetic analogue (LHRHa). This study investigated the impact of low-dose priming injections of LHRHa on gamete-release in the Australian toadlet Pseudophryne guentheri. METHODS: Toadlets were administered a single dose of two micrograms per. gram LHRHa without a priming injection (no priming), or preceded by one (one priming) or two (two priming) injections of 0.4 micrograms per. gram LHRHa. Spermiation responses were evaluated at 3, 7 and 12 hrs post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Oocyte yields were evaluated by stripping females at 10-11 hrs PA. A sub-sample of twenty eggs per female was then fertilised (with sperm obtained from testis macerates) and fertilisation success determined. RESULTS: No priming induced the release of the highest number of spermatozoa, with a step-wise decrease in the number of spermatozoa released in the one and two priming treatments respectively. Peak sperm-release occurred at 12 hrs PA for all priming treatments and there was no significant difference in sperm viability. Females in the control treatment failed to release oocytes, while those administered an ovulatory dose without priming exhibited a poor ovulatory response. The remaining two priming treatments (one and two priming) successfully induced 100% of females to expel an entire clutch. Oocytes obtained from the no, or two priming treatments all failed to fertilise, however oocytes obtained from the one priming treatment displayed an average fertilisation success of 97%. CONCLUSION: Spermiation was most effectively induced in male P. guentheri by administering a single injection of LHRHa without priming. In contrast, female P. guentheri failed to ovulate without priming. A single priming injection induced the release of oocytes of high viability compared to oocytes obtained from females in the two priming treatment which underwent a process of over-ripening.

Human chorionic gonadotropin (a luteinizing hormone homologue) decreases spatial memory and increases brain amyloid-beta levels in female rats.

Numerous studies have suggested that estradiol (E) improves spatial memory as female rats with E perform better than those without E. However there is an inverse relationship between E and luteinizing hormone (LH) levels and LH could play a role. We examined whether treatment with the LH homologue human chorionic gonadotropin (hCG), would impair spatial memory of adult E-treated female rats. In the object location memory task, ovariectomized (ovxed) rats treated with E and either a single high dose (400 IU/kg) or a lower repeated dose of hCG (75 IU/kg hourly for 8 h) showed spatial memory disruption compared to ovxed rats treated with estradiol alone. Impairment was attributed to memory disruption as performance improved with shortened delay between task exposure and testing. Tests on another spatial memory task, the Barnes maze, confirmed that hCG (400 IU/kg) can impair memory: although E+veh treated animals made significantly fewer hole errors across time, E+hCG-treated did not. In humans, high LH levels have been correlated with Alzheimer's disease (AD). Because brain amyloid-beta (Abeta) species have been implicated as a toxic factor thought to cause memory loss in AD, we analyzed whether hCG-treated animals had increased Abeta levels. Levels of Abeta from whole brains or hippocampi were assessed by Western blot. hCG treatment to E-implanted females significantly increased soluble Abeta40 and Abeta42 levels. These results indicate that high levels of LH/hCG can impair spatial memory, and an increase in brain Abeta species may account for the memory impairment in hCG-treated rats.

The contribution of luteinizing hormone to Alzheimer disease pathogenesis.

Several hypotheses have been proposed that attempt to explain the pathogenesis of Alzheimer Disease (AD) including theories involving senile plaque and neurofibrillary tangle formation, increased oxidative stress, and cell cycle abnormalities, since evidence for each of these pathological phenomena have been well documented in AD. Recent epidemiological and experimental data also support a role for the gonadotropin luteinizing hormone in AD. Paralleling the female predominance for developing AD, luteinizing hormone levels are significantly higher in females as compared to males, and furthermore, luteinizing hormone levels are higher still in individuals who succumb to AD. Luteinizing hormone, which is capable of modulating cognitive behavior, is not only present in the brain, but also has the highest receptor levels in the hippocampus, a key processor of cognition that is severely deteriorated in AD. Furthermore, we recently examined cognitive performance in a well-characterized transgenic mouse that over-expresses luteinizing hormone and found that these animals show decreased cognitive performance when compared to controls. We have also found that abolishing luteinizing hormone in amyloid-beta protein precursor transgenic mice (Tg2576) using a potent gonadotropin-lowering gonadotropin-releasing hormone agonist, leuprolide acetate, resulted in improved hippocampally-related cognitive performance and decreased amyloid-beta deposition. These findings, together with data indicating that luteinizing hormone modulates amyloid-beta protein precursor processing in vivo and in vitro, suggest that luteinizing hormone may contribute to AD pathology through an amyloid-dependent mechanism. These promising findings support the importance of luteinizing hormone in AD and bring to the forefront an alternative, and much needed, therapeutic avenue for the treatment of this insidious disease.