Indirect Suppression of Pulsatile LH Secretion by CRH Neurons in the Female Mouse.

Acute stress is a potent suppressor of pulsatile luteinizing hormone (LH) secretion, but the mechanisms through which corticotrophin-releasing hormone (CRH) neurons inhibit gonadotropin-releasing hormone (GnRH) release remain unclear. The activation of paraventricular nucleus (PVN) CRH neurons with Cre-dependent hM3Dq in Crh-Cre female mice resulted in the robust suppression of pulsatile LH secretion. Channelrhodopsin (ChR2)-assisted circuit mapping revealed that PVN CRH neuron projections existed around kisspeptin neurons in the arcuate nucleus (ARN) although many more fibers made close appositions with GnRH neuron distal dendrons in the ventral ARN. Acutely prepared brain slice electrophysiology experiments in GnRH- green fluorescent protein (GFP) mice showed a dose-dependent (30 and 300 nM CRH) activation of firing in ~20% of GnRH neurons in both intact diestrus and ovariectomized mice with inhibitory effects being uncommon (<8%). Confocal GCaMP6 imaging of GnRH neuron distal dendrons in acute para-horizontal brain slices from GnRH-Cre mice injected with Cre-dependent GCaMP6s adeno-associated viruses demonstrated no effects of 30 to 300 nM CRH on GnRH neuron dendron calcium concentrations. Electrophysiological recordings of ARN kisspeptin neurons in Crh-Cre,Kiss1-GFP mice revealed no effects of 30 -300 nM CRH on basal or neurokinin B-stimulated firing rate. Similarly, the optogenetic activation (2-20 Hz) of CRH nerve terminals in the ARN of Crh-Cre,Kiss1-GFP mice injected with Cre-dependent ChR2 had no effect on kisspeptin neuron firing. Together, these studies demonstrate that PVN CRH neurons potently suppress LH pulsatility but do not exert direct inhibitory control over GnRH neurons, at their cell body or dendron, or the ARN kisspeptin neuron pulse generator in the female mouse.

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3.

INTRODUCTION AND OBJECTIVES: SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics. MATERIAL AND METHODS: Different hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl4) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose-response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment. RESULTS: In SerpinB3 transgenic mice, cardiac output (51.6±21.5 vs 30.1±10.8ml/min, p=0.003), hepatic artery pulsatility index (0.85±0.13 vs 0.65±0.11, p<0.001) and portal vein blood flow (5.3±3.2 vs 3.1±1.8ml/min, p=0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p<0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors. CONCLUSIONS: In transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.

Cerebrovascular reactivity after cessation of menopausal hormone treatment.

OBJECTIVE: Women who are currently using menopausal hormone therapy (MHT) have higher cerebrovascular reactivity when compared with postmenopausal women who are not taking MHT; however, the effect of cessation of MHT on cerebrovascular reactivity is not known. Given that MHT can have structural and activational effects on vascular function, this study was performed to characterize cerebrovascular reactivity following cessation of MHT in women at low risk for cerebrovascular disease. METHODS: Cerebrovascular reactivity was measured in a subset of women from the Kronos Early Estrogen Prevention Study (KEEPS) 3 years after cessation of the study drug (oral conjugated equine estrogen, transdermal 17ß-estradiol, or placebo [PLA]). RESULTS: Age, body mass index, and blood pressure were comparable among groups. At rest, the middle cerebral artery velocity (MCAv), cerebrovascular conductance index, mean arterial pressure, and cerebral pulsatility index did not differ among groups. Slope-based summary measures of cerebrovascular reactivity did not differ significantly among groups. However, utilizing repeated-measures modeling, there was a significant upward shift in MCAv responses (p = 0.029) in the combined MHT group compared with the PLA group. CONCLUSION: MHT has a marginal sustained effect on cerebrovascular reactivity when measured 3 years after cessation of hormone treatment.

Non-pulsatile blood flow is associated with enhanced cerebrovascular carbon dioxide reactivity and an attenuated relationship between cerebral blood flow and regional brain oxygenation.

BACKGROUND: Systemic blood flow in patients on extracorporeal assist devices is frequently not or only minimally pulsatile. Loss of pulsatile brain perfusion, however, has been implicated in neurological complications. Furthermore, the adverse effects of absent pulsatility on the cerebral microcirculation are modulated similarly as CO2 vasoreactivity in resistance vessels. During support with an extracorporeal assist device swings in arterial carbon dioxide partial pressures (PaCO2) that determine cerebral oxygen delivery are not uncommon-especially when CO2 is eliminated by the respirator as well as via the gas exchanger of an extracorporeal membrane oxygenation machine. We, therefore, investigated whether non-pulsatile flow affects cerebrovascular CO2 reactivity (CVR) and regional brain oxygenation (rSO2). METHODS: In this prospective, single-centre case-control trial, we studied 32 patients undergoing elective cardiac surgery. Blood flow velocity in the middle cerebral artery (MCAv) as well as rSO2 was determined during step changes of PaCO2 between 30, 40, and 50 mmHg. Measurements were conducted on cardiopulmonary bypass during non-pulsatile and postoperatively under pulsatile blood flow at comparable test conditions. Corresponding changes of CVR and concomitant rSO2 alterations were determined for each flow mode. Each patient served as her own control. RESULTS: MCAv was generally lower during hypocapnia than during normocapnia and hypercapnia (p < 0.0001). However, the MCAv/PaCO2 slope during non-pulsatile flow was 14.4 cm/s/mmHg [CI 11.8-16.9] and 10.4 cm/s/mmHg [CI 7.9-13.0] after return of pulsatility (p = 0.03). During hypocapnia, non-pulsatile CVR (4.3 ± 1.7%/mmHg) was higher than pulsatile CVR (3.1 ± 1.3%/mmHg, p = 0.01). Independent of the flow mode, we observed a decline in rSO2 during hypocapnia and a corresponding rise during hypercapnia (p < 0.0001). However, the relationship between ΔrSO2 and ΔMCAv was less pronounced during non-pulsatile flow. CONCLUSIONS: Non-pulsatile perfusion is associated with enhanced cerebrovascular CVR resulting in greater relative decreases of cerebral blood flow during hypocapnia. Heterogenic microvascular perfusion may account for the attenuated ΔrSO2/ΔMCAv slope. Potential hazards related to this altered regulation of cerebral perfusion still need to be assessed. TRIAL REGISTRATION: The study was retrospectively registered on October 30, 2018, with Clinical Trial.gov (NCT03732651).

Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury.

BACKGROUND: Arterial stiffness is emerging as an independent risk factor for the development of chronic kidney disease. The sodium glucose co-transporter 2 (SGLT2) inhibitors, which lower serum glucose by inhibiting SGLT2-mediated glucose reabsorption in renal proximal tubules, have shown promise in reducing arterial stiffness and the risk of cardiovascular and kidney disease in individuals with type 2 diabetes mellitus. Since hyperglycemia contributes to arterial stiffness, we hypothesized that the SGLT2 inhibitor empagliflozin (EMPA) would improve endothelial function, reduce aortic stiffness, and attenuate kidney disease by lowering hyperglycemia in type 2 diabetic female mice (db/db). MATERIALS/METHODS: Ten-week-old female wild-type control (C57BLKS/J) and db/db (BKS.Cg-Dock7m+/+Leprdb/J) mice were divided into three groups: lean untreated controls (CkC, n = 17), untreated db/db (DbC, n = 19) and EMPA-treated db/db mice (DbE, n = 19). EMPA was mixed with normal mouse chow at a concentration to deliver 10 mg kg-1 day-1, and fed for 5 weeks, initiated at 11 weeks of age. RESULTS: Compared to CkC, DbC showed increased glucose levels, blood pressure, aortic and endothelial cell stiffness, and impaired endothelium-dependent vasorelaxation. Furthermore, DbC exhibited impaired activation of endothelial nitric oxide synthase, increased renal resistivity and pulsatility indexes, enhanced renal expression of advanced glycation end products, and periarterial and tubulointerstitial fibrosis. EMPA promoted glycosuria and blunted these vascular and renal impairments, without affecting increases in blood pressure. In addition, expression of "reversion inducing cysteine rich protein with Kazal motifs" (RECK), an anti-fibrotic mediator, was significantly suppressed in DbC kidneys and partially restored by EMPA. Confirming the in vivo data, EMPA reversed high glucose-induced RECK suppression in human proximal tubule cells. CONCLUSIONS: Empagliflozin ameliorates kidney injury in type 2 diabetic female mice by promoting glycosuria, and possibly by reducing systemic and renal artery stiffness, and reversing RECK suppression.

Uterine artery flow velocity waveform, arterial flow indices, follicular dynamics, and sex hormones during preovulatory period in synchronized ovulatory cycle of Bos indicus beef cows.

A greater understanding of the uterine artery's (UtA) biology is essential to the increase in female reproductive abilities. The UtA flow velocity waveform, blood flow volume (BFV), pulsatility and resistance indices (PI and RI), blood flow velocities, dynamics of the dominant follicle (DF), and estradiol (E2) and progesterone (P4) levels in an induced ovulatory cycle were evaluated in Thai native cattle. Twenty cows were induced with synchronized ovulation through a P4-releasing device, from Day -9 to Day -4, concurrent with the administration of two doses of a gonadotropin-releasing hormone on Day -9 and Day -1, and two doses of prostaglandin F2α on Day -4 and 8 h later. Day 0 was designated as the day of ovulation. The cows underwent Doppler sonographic determination and blood collection from Day -4 to Day 0. The cows were classified in the non-ovulating (n = 5) and ovulating groups (n = 15). The ovulating cows presented higher BFV values, blood flow velocities, DF growth rates, and E2 levels; yet lower PI values and P4 concentrations, than those of the non-ovulating cows. The BFV values and the blood flow velocities were greater, but the RI and PI values were lower in the ovulatory side UtA than in the contraovulatory side UtA. The BFV values were positively correlated with blood flow velocities, DF growth rates and E2 concentrations in the ovulating cows; confirming the importance of UtA blood flow, follicular growth, and E2-vasodilation during preovulatory phase in the induced ovulatory cycle of Bos indicus beef cows.

Progesterone Suppression of Luteinizing Hormone Pulse Frequency in Adolescent Girls With Hyperandrogenism: Effects of Metformin.

Context: Polycystic ovary syndrome (PCOS) and adolescent hyperandrogenism (HA) are characterized by rapid luteinizing hormone (LH) pulse frequency. This partly reflects impaired gonadotropin-releasing hormone pulse generator (hypothalamic) sensitivity to progesterone (P4) negative feedback. We assessed whether metformin may improve P4 sensitivity in adolescent HA, for which it is prescribed widely. Objective: To test the hypothesis that metformin improves hypothalamic P4 sensitivity in adolescent HA. Design: Nonrandomized, interventional trial. Setting: Academic clinical research unit. Participants: Ten adolescent girls with HA. Intervention: The girls underwent LH sampling every 10 minutes for 11 hours, at study baseline and after 7 days of oral P4 and estradiol (E2). Participants then took metformin (1 g twice daily) for 9.4 to 13.7 weeks, after which participants again underwent frequent LH sampling before and after 7 days of oral P4 and E2 (while continuing metformin). Total and free testosterone (T) and fasting insulin were assessed at each admission. At admissions 1 and 3, 2-hour oral glucose tolerance tests were performed. Main Outcome Measure: Metformin-related change in hypothalamic P4 sensitivity index [percent change in LH pulse frequency (before vs after P4 and E2) divided by day 7 P4 level]. Results: Free T levels decreased by 29% with metformin (P = 0.0137). Measures of hyperinsulinemia and P4 sensitivity index did not significantly change with metformin use. Conclusion: Short-term metformin use improved biochemical hyperandrogenemia, but did not improve hypothalamic sensitivity to P4 suppression, in adolescent girls.

Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats.

BACKGROUND: Disturbances in coronary microcirculatory function, such as the endothelial glycocalyx, are early hallmarks in the development of obesity and insulin resistance. Accordingly, in the present study myocardial microcirculatory perfusion during rest and stress was assessed following metformin or sulodexide therapy in a rat model of diet-induced obesity. Additionally, the effect of degradation of the glycocalyx on myocardial perfusion was assessed in chow-fed rats. METHODS: Rats were fed a high fat diet (HFD) for 8 weeks and were divided into a group without therapy, and groups that received the anti-diabetic drug metformin or the glycocalyx-stabilizing drug sulodexide in their drinking water during the last 4 weeks of the feeding period. Myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle. In an acute experimental setting, hyaluronidase was administered to chow-fed control rats to determine the effect of enzymatical degradation of the glycocalyx on myocardial perfusion. RESULTS: HFD-rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. We confirmed our earlier findings that the robust increase in myocardial perfusion in chow-fed rats after an adenosine challenge (+56%, p = 0.002) is blunted in HFD rats (+8%, p = 0.68). In contrast, 4-weeks treatment with metformin or sulodexide partly restored the increase in myocardial perfusion during adenosine infusion in HFD rats (+81%, p = 0.002 and +37%, p = 0.02, respectively). Treating chow-fed rats acutely with hyaluronidase, to enzymatically degrade the glyocalyx, completely blunted the increase in myocardial perfusion during stress. CONCLUSIONS: In early stages of HFD-induced insulin resistance myocardial perfusion becomes compromised, a process that can be countered by treatment with both metformin and sulodexide. The adverse effect of acute glycocalyx degradation and protective effect of long-term sulodexide administration on myocardial perfusion provides indirect evidence, suggesting a role for the glycocalyx in preserving coronary microvascular function in pre-diabetic animals.

Estrogen Preserves Pulsatile Pulmonary Arterial Hemodynamics in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is caused by extensive pulmonary vascular remodeling that increases right ventricular (RV) afterload and leads to RV failure. PAH predominantly affects women; paradoxically, female PAH patients have better outcomes than men. The roles of estrogen in PAH remain controversial, which is referred to as "the estrogen paradox". Here, we sought to determine the role of estrogen in pulsatile pulmonary arterial hemodynamic changes and its impact on RV functional adaption to PAH. Female mice were ovariectomized and replenished with estrogen or placebo. PAH was induced with SU5416 and chronic hypoxia. In vivo hemodynamic measurements showed that (1) estrogen prevented loss of pulmonary vascular compliance with limited effects on the increase of pulmonary vascular resistance in PAH; (2) estrogen attenuated increases in wave reflections in PAH and limited its adverse effects on PA systolic and pulse pressures; and (3) estrogen maintained the total hydraulic power and preserved transpulmonary vascular efficiency in PAH. This study demonstrates that estrogen preserves pulmonary vascular compliance independent of pulmonary vascular resistance, which provides a mechanical mechanism for ability of estrogen to delay disease progression without preventing onset. The estrogenic protection of pulsatile pulmonary hemodynamics underscores the therapeutic potential of estrogen in PAH.

Progesterone has rapid positive feedback actions on LH release but fails to reduce LH pulse frequency within 12 h in estradiol-pretreated women.

In women, progesterone suppresses luteinizing hormone (LH) (gonadotropin-releasing hormone) pulse frequency, but how rapidly this occurs is unknown. In estradiol-pretreated women in the late follicular phase, progesterone administration at 1800 did not slow sleep-associated LH pulse frequency. However, mechanisms controlling LH pulse frequency may differ according to sleep status; and we thus hypothesized that progesterone acutely suppresses waking LH pulse frequency. This was a randomized, double-blind, crossover study of LH secretory responses to progesterone versus placebo administered at 0600. We studied 12 normal women in the late follicular phase (cycle days 7-11), pretreated with 3 days of transdermal estradiol (0.2 mg/day). Subjects underwent a 24-h blood sampling protocol (starting at 2000) and received either 100 mg oral micronized progesterone or placebo at 0600. In a subsequent menstrual cycle, subjects underwent an identical protocol except that oral progesterone was exchanged for placebo or vice versa. Changes in 10-h LH pulse frequency were similar between progesterone and placebo. However, mean LH, LH pulse amplitude, and mean follicle-stimulating hormone exhibited significantly greater increases with progesterone. Compared to our previous study (progesterone administered at 1800), progesterone administration at 0600 was associated with a similar increase in mean LH, but a less pronounced increase in LH pulse amplitude. We conclude that, in estradiol-pretreated women in the late follicular phase, a single dose of progesterone does not suppress waking LH pulse frequency within 12 h, but it acutely amplifies mean LH and LH pulse amplitude - an effect that may be influenced by sleep status and/or time of day.

Middle Cerebral Artery Pulsatility Index is Associated with Cognitive Impairment in Lacunar Stroke.

BACKGROUND AND PURPOSE: Pulsatility index (PI) of the middle cerebral artery is postulated to reflect the vascular resistance in the artery distal of the probe, and has been reported to increase in small vessel disease, diabetes mellitus, ageing, and dementia. Lacunar infarcts are considered to be related to cognitive impairment. We therefore conducted a study to assess the association between cognitive impairment and PI in patients with a lacunar infarct. METHODS: Consecutive patients presenting with an acute lacunar syndrome who were admitted to the stroke unit were enrolled. The patients were examined with Doppler ultrasonography of the intracranial arteries, and the PI of the middle cerebral artery was recorded. Cognitive function was evaluated by mini-mental state examination (MMSE), clock drawing test, and trail making test (TMT) A and B. RESULTS: Among the 113 patients included, 85 patients had an acute lacunar infarct and 28 had one or more nonlacunar infarcts. The mean PI was 1.46 (SD = .33). PI was significantly (P < .05) associated with MMSE, TMT A and TMT B in patients with lacunar infarct, even after adjustment for multiple patient characteristics (age, sex, prestroke hypertension, smoking, previous stroke, and diabetes). CONCLUSIONS: PI was associated with the cognitive performance in patients with lacunar infarcts and a lacunar syndrome. An elevated PI may be related to impairment in several cognitive domains. These findings suggest that transcranial Doppler ultrasonography could be an adjunct tool for early diagnosis of cognitive impairment after stroke.

Increasing LH pulsatility in women with hypothalamic amenorrhoea using intravenous infusion of Kisspeptin-54.

BACKGROUND: Hypothalamic amenorrhea (HA) is the one of the most common causes of period loss in women of reproductive age and is associated with deficient LH pulsatility. High-dose kisspeptin-54 acutely stimulates LH secretion in women with HA, but chronic administration causes desensitization. GnRH has paradoxical effects on reproductive activity; we therefore hypothesized that a dose-dependent therapeutic window exists within which kisspeptin treatment restores the GnRH/LH pulsatility in women with HA. AIM: The aim of the study was to determine whether constant iv infusion of kisspeptin-54 temporarily increases pulsatile LH secretion in women with HA. METHODS: Five patients with HA each underwent six assessments of LH pulsatility. Single-blinded continuous iv infusion of vehicle or kisspeptin-54 (0.01, 0.03, 0.10, 0.30, or 1.00 nmol/kg/h) was administered. The LH pulses were detected using blinded deconvolution. RESULTS: Kisspeptin increased LH pulsatility in all patients with HA, with peak responses observed at different doses in each patient. The mean peak number of pulses during infusion of kisspeptin-54 was 3-fold higher when compared with vehicle (number of LH pulses per 8 h: 1.6 ± 0.4, vehicle; 5.0 ± 0.5, kisspeptin-54, P < .01 vs vehicle). The mean peak LH pulse secretory mass during kisspeptin-54 was 6-fold higher when compared with vehicle (LH pulse secretory mass in international units per liter: 3.92 ± 2.31, vehicle; 23.44 ± 12.59, kisspeptin-54; P < .05 vs vehicle). CONCLUSIONS: Kisspeptin-54 infusion temporarily increases LH pulsatility in women with HA. Furthermore, we have determined the dose range within which kisspeptin-54 treatment increases basal and pulsatile LH secretion in women with HA. This work provides a basis for studying the potential of kisspeptin-based therapies to treat women with HA.

Estradiol regulates GH-releasing peptide's interactions with GH-releasing hormone and somatostatin in postmenopausal women.

OBJECTIVE: Estrogen stimulates pulsatile secretion of GH, via mechanisms that are largely unknown. An untested hypothesis is that estradiol (E2) drives GH secretion by amplifying interactions among GH-releasing hormone (GHRH), somatostatin (SS), and GH-releasing peptide (GHRP). DESIGN: The design comprised double-blind randomized prospective administration of transdermal E2 vs placebo to healthy postmenopausal women (n=24) followed by pulsatile GHRH or SS infusions for 13 h overnight with or without continuous GHRP2 stimulation. METHODS: End points were mean concentrations, deconvolved secretion, and approximate entropy (ApEn; a regularity measure) of GH. RESULTS: By generalized ANOVA models, it was observed that E2 vs placebo supplementation: i) augmented mean (13-h) GH concentrations (P=0.023), GHRH-induced pulsatile GH secretion over the first 3 h (P=0.0085) and pulsatile GH secretion over the next 10 h (P=0.054); ii) increased GHRP-modulated (P=0.022) and SS-modulated (P<0.001) GH ApEn; and iii) did not amplify GHRH/GHRP synergy during pulsatile GH secretion. By linear regression, E2 concentrations were found to be positively correlated with GH secretion during GHRP2 infusion (P=0.022), whereas BMI was found to be negatively correlated with GH secretion during GHRH (P=0.006) and combined GHRH/GHRP (P=0.015) stimulation. E2 and BMI jointly determined triple (combined l-arginine, GHRH, and GHRP2) stimulation of GH secretion after saline (R²=0.44 and P=0.003) and pulsatile GHRH (R²=0.39 and P=0.013) infusions. CONCLUSION: In summary, in postmenopausal women, E2 supplementation augments the amount (mass) and alters the pattern (regularity) of GH secretion via interactions among GHRH, SS, GHRP, and BMI. These outcomes introduce a more complex model of E2 supplementation in coordinating GH secretion in aging women.

[Influence of iron nanoparticles on cardiac performance and hemodynamics in rabbits after intravenous administration in acute experiment].

Iron nanoparticles are possessed by high potential in the creation of effective and safe antianemic drugs due to the enhanced biological activity of metal nanoparticles. As a step of intravenous dosage form development the study of short-term effects of iron nanoparticles on the cardiovascular system is important. Dose-dependent changes of systemic hemodynamics' parameters were established in acute experiment on rabbits after several intravenous injections of zero-valent iron nanoparticles solution.

A single injection of kisspeptin-54 temporarily increases luteinizing hormone pulsatility in healthy women.

BACKGROUND: Kisspeptin is a novel hypothalamic peptide which stimulates endogenous gonadotrophin releasing hormone (GnRH) secretion. A single subcutaneous bolus injection of kisspeptin-54 increases circulating luteinizing hormone (LH) levels in women, but its acute effects on LH pulsatility are not known. AIMS: To investigate the effects of a single subcutaneous (sc) injection of kisspeptin-54 administration on LH pulsatility in healthy female volunteers. METHODS: Six healthy female adult volunteers underwent 10-minute blood sampling for serum LH measurement for 8 h during the follicular phase of menstrual cycle. Sc bolus injection of saline or kisspeptin-54 (0·15, 0·30 or 0·60 nmol/kg) was administered 4 h after commencing the study. A previously described, blinded deconvolution method was used to detect LH pulses. RESULTS: Mean number of LH pulses was increased significantly following 0·30 and 0·60 nmol/kg kisspeptin-54 when compared with saline (mean increase in number of LH pulses per 4 h, following injection: -0·17 ± 0·54, saline; +2·33 ± 0·56, 0·30 nmol/kg kisspeptin-54, P < 0·05 vs saline; +2·33 ± 0·80, 0·60 nmol/kg kisspeptin-54, P < 0·05 vs saline). LH pulse secretory mass increased following injection of 0·60 nmol/kg in five of six subjects, but the mean change in all subjects was non-significant when compared with saline (mean increase in pulse secretory mass in IU/l following injection: +0·35 ± 0·40, saline; +2·61 ± 1·17, 0·60 nmol/kg kisspeptin-54, P = 0·10 vs saline). CONCLUSIONS: A single injection of kisspeptin-54 temporarily stimulates the number of LH pulses in healthy women. Further studies are required to investigate the therapeutic potential of kisspeptin-54 injection to restore LH pulsatility in patients with reproductive disorders caused by impaired GnRH secretion.

Doppler of the uterine arteries combined with endometrial thickness correlate well with the degree of pituitary suppression in women treated with long-acting GnRH agonists.

PURPOSE: The aim of this study was to evaluate the use of Doppler velocimetry of the uterine arteries and its association to endometrial thickness as a method to confirm pituitary suppression after administration of gonadotropin-releasing hormone analogues in assisted reproduction treatment cycles. METHODS: A total of 70 patients using gonadotropin-releasing hormone analogues for pituitary suppression for in vitro fertilization treatment were studied. To confirm down-regulation, serum estradiol levels and endometrial thickness were evaluated 10 days after gonadotropin-releasing hormone analogues administration. When estradiol was <30 pg/ml and endometrial thickness was <3 mm, pituitary suppression was confirmed. Doppler velocimetric measurements were performed at the same day to study the pulsatility index of the uterine arteries, until pituitary suppression was confirmed. RESULTS: All 70 patients had normal ovarian morphology. For the patients who had estradiol levels ≤30 pg/ml, the mean pulsatility index of the uterine arteries was 2.95 ± 0.79 and for those who had levels >30 pg/ml the mean PI was 2.22 ± 0.8 (p = 0.005). For the patients who had endometrial thickness ≤5 mm the mean PI was 2.86 ± 0.82 and for those with endometrial thickness >5 mm the mean PI was 2.17 ± 0.79 (p = 0.004). Using a cut-off point of 2.51 for the pulsatility index, to compare to estradiol levels, we observed a sensitivity of 72.7 % and specificity of 71 %. The combination of Doppler velocimetric and endometrial thickness showed a sensitivity of 94 % and specificity of 82.3 %. CONCLUSIONS: Doppler velocimetric analysis of the uterine arteries can be an important tool in the diagnosis of the down-regulation after the use of gonadotropin-releasing hormone analogues and might help simplify assisted reproduction programmes.

Kisspeptin restores pulsatile LH secretion in patients with neurokinin B signaling deficiencies: physiological, pathophysiological and therapeutic implications.

Pulsatile gonadotropin-releasing hormone (GnRH) is crucial to normal reproductive function and abnormalities in pulse frequency give rise to reproductive dysfunction. Kisspeptin and neurokinin B (NKB), neuropeptides secreted by the same neuronal population in the ventral hypothalamus, have emerged recently as critical central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We report here that continuous kisspeptin infusion restores gonadotropin pulsatility in patients with loss-of-function mutations in NKB (TAC3) or its receptor (TAC3R), indicating that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin.

Effects of lack of pulsatility on pulmonary endothelial function in the Fontan circulation.

OBJECTIVES: Continuous flow in the Fontan circulation results in impairment of pulmonary artery endothelial function, increased pulmonary arterial resistance, and, potentially, late failure of Fontan circulation. We investigated the mechanisms of vascular remodeling and altered vascular reactivity associated with chronic privation of pulsatility on pulmonary vasculature. METHODS: A total of 30 pigs were evenly distributed in 3 groups: 10 underwent a sham procedure (group I) and 20 underwent a cavopulmonary shunt between the superior vena cava and right pulmonary artery--10 with complete ligation of the proximal right pulmonary artery (group II, nonpulsatile) and 10 with partial ligation (group III, micropulsatile). At 3 months postoperatively, the in vivo hemodynamics, in vitro vasomotricity (concentration response curves on pulmonary artery isolated rings), and endothelial nitric oxide synthase protein level were assessed. A comparison between group and between the right and left lung in each group was performed. RESULTS: Group II developed right pulmonary hypertension and increased right pulmonary resistance. Endothelial function was altered in group II, as reflected by a decrease in the vasodilation response to acetylcholine and ionophoric calcium but preservation of the nonendothelial-dependent response to sodium nitroprusside. Group III micropulsatility attenuated pulmonary hypertension but did not prevent impairment of the endothelial-dependant relaxation response. Right lung Western blotting revealed decreased endothelial nitric oxide synthase in group II (0.941 ± 0.149 vs sham 1.536 ± 0.222, P = .045) that was preserved in group III (1.275 ± 0.236, P = .39). CONCLUSIONS: In a chronic model of unilateral cavopulmonary shunt, pulsatility loss resulted in an altered endothelial-dependant vasorelaxation response of the pulmonary arteries. Micropulsatility limited the effects of pulsatility loss. These results are of importance for potential therapies against pulmonary hypertension in the nonpulsatile Fontan circulation, by retaining accessory pulmonary flow or pharmaceutical modulation of nonendothelial-dependant pulmonary vasorelaxation.

Temporal interrelationships at 15-min intervals among oxytocin, LH, and progesterone during a pulse of a prostaglandin F2α metabolite in heifers.

A pulse of a PGF2α metabolite (PGFM) was induced by treatment with 0.1 mg of estradiol-17ß on Day 15 (Day 0=ovulation; n=9 heifers). Blood samples were taken every 15 min for 9h beginning at treatment (Hour 0). For PGFM and LH, an intraassay-CV method was used to detect fluctuations in the 15-min samples and pulses in the hourly samples. A mean of 6.9 ± 0.4 PGFM fluctuations/9 h were superimposed on the hourly PGFM concentrations, compared to 2.1 ± 0.5 LH fluctuations/9 h (P<0.02). An increase (P<0.02) in oxytocin began 15 min before the beginning nadir of the PGFM pulse. A transient increase in progesterone did not occur at the beginning nadir of the PGFM pulse. Progesterone decreased (P<0.02) during the ascending portion and increased (P<0.03) as a rebound during the descending portion of the PGFM pulse. The peak of an LH pulse occurred 1.5 ± 0.4 h (range, 0.25-2.75 h) after the peak of the PGFM pulse. The wide range in the interval from a PGFM peak to an LH peak obscured the contribution of increasing LH to the rebound. The results did not support the hypothesis that oxytocin and PGFM increase concurrently. Results supported the hypothesis that the immediate transient progesterone increase that has been demonstrated with exogenous PGF2α does not occur during the ascending portion of an endogenous PGFM pulse. The hypothesis that the progesterone rebound after the peak of a PGFM pulse is temporally related to an LH pulse was supported.

Effect of letrozole on uterine artery Doppler flow indices prior to first-trimester termination of pregnancy: a randomized controlled trial.

OBJECTIVE: We previously demonstrated that a sequential regimen of letrozole and misoprostol resulted in a marked reduction in the serum estradiol concentration and in a higher efficacy of first-trimester termination of pregnancy than misoprostol alone. The aim of this study was to evaluate the effect of letrozole on uterine artery Doppler flow indices during early pregnancy. METHODS: This was a randomized controlled trial. Thirty women requesting termination of pregnancy up to 63 days' gestation were randomized into two groups: a letrozole group receiving 10 mg of letrozole, daily, for 3 days, and a control group receiving a placebo for 3 days. Serum estradiol, progesterone and human chorionic gonadotropin (hCG) concentrations were measured before drug administration and then daily for 6 days. Ultrasound scanning for fetal viability and measurement of the pulsatility (PI) and resistance (RI) indices of the uterine arteries was performed before drug administration, and then on day 3 and day 7 after starting letrozole or placebo. All pregnancies were terminated by surgical evacuation on day 7 or day 8. RESULTS: Uterine artery PI and RI decreased significantly in the letrozole group, but not in the control group. Serum estradiol concentrations were significantly lower in the letrozole group than in the control group from day 2 onwards. Serum progesterone and hCG concentrations were comparable for the two groups throughout the 7 days. There were significantly more women in the letrozole group with vaginal bleeding. CONCLUSION: We have demonstrated that the use of letrozole in the first trimester of pregnancy suppresses serum estradiol levels but results in an increase in blood flow to the uterus. Further studies should be carried out to elucidate the mechanism of letrozole pretreatment in medical abortion.