The effect of morbid obesity or advanced maternal age on oxytocin-induced myometrial contractions: an in vitro study.

PURPOSE: The purpose of this study was to investigate myometrial contractility induced by oxytocin in women with advanced maternal age (AMA) and morbid obesity (MO). We hypothesized that both oxytocin-pretreated and oxytocin-naïve myometrial tissues from women of AMA and women who are MO would exhibit poor myometrial contractility compared with women that are younger and of normal body mass index (BMI). METHODS: This prospective in vitro study was conducted using myometrial samples obtained from women undergoing elective Cesarean deliveries. Three groups of patient were studied: control (≤ 35 yr; BMI, 20-24.9 kg·m-2), AMA (≥ 40 yr; BMI, 20-24.9 kg·m-2), and MO (≤ 35 yr BMI, ≥ 40 kg·m-2). Each myometrial strip was either pretreated with oxytocin 10-5 M or left in physiologic salt solution for two hours. This was followed by a dose-response testing to oxytocin (10-10 M to 10-5 M), during which contractile parameters were measured. The primary outcome was motility index (MI, amplitude × frequency) of contractions. RESULTS: The MI of contractions was reduced in oxytocin-pretreated samples when compared with their oxytocin naïve counterparts in control (estimated difference -69%; 95% confidence interval [CI], -82 to -48; P < 0.001) and AMA groups (estimated difference, -44%; 95% CI, -68 to -2; P = 0.07). The MI of contractions was not different between oxytocin naïve and oxytocin-pretreated samples from MO women (estimated difference, -26%; 95% CI, -63 to 49; P = 0.46); however, it was significantly lower in these groups compared with oxytocin-naïve samples from the control group. CONCLUSIONS: Oxytocin pre-treatment reduced myometrial contractility in AMA and control group women compared with their oxytocin-naïve counterparts, as a function of the desensitization phenomenon. Attenuated oxytocin-induced myometrial contractility in MO women in both oxytocin-pretreated and oxytocin-naïve samples suggests that these women have intrinsically reduced uterine contractile ability. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT01865669; registered 28 May, 2013).

RéSUMé: OBJECTIF: L'objectif de cette recherche était d'étudier la contractilité myométriale induite par l'oxytocine chez des femmes d'âge maternel avancé (AMA) et ayant une obésité morbide (OM). Nous avons formulé l'hypothèse que les tissus de myomètre prétraités avec de l'oxytocine ainsi que les tissus naïfs d'oxytocine provenant de femmes d'AMA ou ayant une OM présenteraient une mauvaise contractilité comparativement à des femmes plus jeunes et à l'indice de masse corporelle (IMC) normal. MéTHODES: Cette étude prospective in vitro a été menée en utilisant des échantillons de myomètre prélevés sur des femmes accouchant par césarienne planifiée. Trois groupes de patientes ont été étudiés : contrôles (≤ 35 ans; IMC, 20 à 24,9 kg·m−2), AMA (≥ 40 ans; IMC, 20 à 24,9 kg·m−2) et OM (≤ 35 ans IMC, ≥ 40 kg·m−2). Chaque bandelette de myomètre a été prétraitée avec de l'oxytocine 10−5 M ou laissée dans une solution salée physiologique pendant deux heures. Cela a été suivi de tests de réponse à des doses croissantes d'oxytocine (10−10 M à 10−5 M) au cours desquels les paramètres contractiles ont été mesurés. Le critère d'évaluation principal était l'indice de motilité (IM, amplitude × fréquence) des contractions. RéSULTATS: Cet IM des contractions était diminué dans les échantillons prétraités avec l'oxytocine, comparativement à leurs équivalents naïfs d'oxytocine dans les groupes contrôles (différence estimée -69 %; intervalle de confiance [IC] à 95 % : -82 à -48; P < 0,001) et AMA (différence estimée, -44 %; IC à 95 %, -68 à -2; P = 0,07). L'IM des contractions n'était pas différent entre les échantillons naïfs d'oxytocine et les échantillons prétraités provenant de femmes ayant une OM (différence estimée, -26 %; IC à 95 %, -63 à 49; P = 0,46). Il était cependant significativement plus faible dans ces groupes comparativement aux échantillons naïfs pour l'oxytocine provenant du groupe contrôle. CONCLUSIONS: Le prétraitement à l'oxytocine a réduit la contractilité myométriale des femmes des groupes AMA et contrôle comparativement à celle des prélèvements naïfs d'oxytocine en fonction d'un phénomène de désensibilisation. L'atténuation de la contractilité myométriale induite par l'oxytocine dans les échantillons prétraités et naïfs d'oxytocine chez les femmes présentant une OM suggère que ces dernières présentent une capacité contractile intrinsèquement réduite de l'utérus. ENREGISTREMENT DE L'ESSAI CLINIQUE: www.clinicaltrials.gov (NCT01865669; enregistré le 28 mai 2013).

Outpatient High-dose Methotrexate for Osteosarcoma: It's Safe and Feasible, If You Want It.

BACKGROUND: High-dose methotrexate (HD MTX) is usually administered as an inpatient to those with osteosarcoma. We prospectively tested the safety and feasibility of administering HD MTX in the ambulatory setting. MATERIALS AND METHODS: In this single arm prospective observational study, eligible patients had previously completed 2 courses of HD MTX as an inpatient. On study, patients received MTX in hospital, discharged home and returned for daily assessment. Criteria to determine safety and feasibility included: (1) parent compliance with home instructions, (2) pump functioning/failure, and/or (3) admission for toxicity/noncompliance. Outpatient therapy was deemed feasible if <25% courses resulted in study event. Patient satisfaction was assessed. RESULTS: Six patients (median age, 13.5 y) with extremity osteosarcoma completed 35 courses of MTX. There were no study events-no hospitalizations or pump failures and all parents were compliant. The Data and Safety Committee concluded that with zero events in 35 courses, it was unlikely for outpatient MTX to be infeasible; study was thus terminated early. Participants reported value to stay out of hospital, permitted life to feel "more normal"; however, burden of daily commute to hospital was cited. CONCLUSIONS: The delivery of HD MTX is safe and feasible in patients with osteosarcoma.

Pediatric oncology clinical trial participation where the geography is vast: Development of a clinical research system for tertiary and satellite centers in Ontario, Canada.

Opportunities for participation in clinical trials are a core component of the care of children with cancer. In Ontario, many pediatric patients live long distances from their cancer center. This paper describes the work that was done in order to allow patients participating in Children's Oncology Group trials to receive care, including research protocol related care, jointly between the tertiary pediatric cancer center and the closer-to-home satellite center. The system is a pragmatic risk-based model, supporting excellence in care while ensuring good conduct of the research in compliance with applicable regulations and guidelines, including ethics oversight.

The Recovery Time of Myometrial Responsiveness After Oxytocin-Induced Desensitization in Human Myometrium In Vitro.

BACKGROUND: Postpartum hemorrhage secondary to uterine atony is a leading cause of maternal morbidity. Prolonged exposure to oxytocin for labor augmentation can result in the desensitization phenomenon, a decrease in the responsiveness of myometrium to further oxytocin. It is currently not known whether waiting for a specific time interval after the cessation of oxytocin allows the oxytocin receptors to resensitize and recover, thereby improving subsequent oxytocin-induced myometrial contractility. We aimed to investigate the effect of a rest period of 30, 60, and 90 minutes after oxytocin administration on the recovery of oxytocin-desensitized human myometrium in vitro. We hypothesized that the longer the rest period, the better the responsiveness and subsequent oxytocin-induced contractility of the myometrium. METHODS: Myometrial tissue was obtained from women undergoing elective cesarean deliveries. The myometrial sample was dissected into 4 strips, and each strip was mounted in a single organ bath with physiological salt solution (PSS) under homeostatic conditions and then pretreated for 2 hours with oxytocin 10 M. After pretreatment, each strip was washed with PSS and allowed to rest in PSS solution for 30, 60, or 90 minutes. At the end of the rest period, dose-response testing to oxytocin 10 to 10 M was performed. A control group consisted of oxytocin dose-response testing without any oxytocin pretreatment. Contractile parameters were measured and compared among the groups after square root transformation. The primary outcome was motility index (frequency × amplitude), and secondary outcomes included frequency, amplitude, and area under the curve. RESULTS: Fifty-five experiments were conducted from samples obtained from 16 women. The mean motility index (√g·contractions/10 min) during the dose-response curve (oxytocin 10 to 10 M) in the control group was significantly greater than all the experimental groups; the mean estimated differences (95% confidence intervals) were -1.33 (-2.50 to -0.15, P = 0.02), -1.59 (-2.68 to -0.50, P = 0.004), and -1.88 (-2.97 to -0.80, P = 0.001) for the 30-, 60-, and 90-minute groups, respectively. When the experimental groups were compared, there were no significant differences in any of the contractility parameters; however, confidence intervals were wide. CONCLUSIONS: Our study shows that oxytocin pretreatment attenuates oxytocin-induced contractility in human myometrium despite a rest period of up to 90 minutes after oxytocin administration. However, we were unable to determine whether increasing the rest period from 30 to 90 minutes results in improvement in myometrial contractility because of our small sample size relative to the variability in the contractile parameters. Further laboratory and clinical in vivo studies are necessary to determine whether a rest period up to 90 minutes results in improvement in myometrial contractility. In addition, further experimental studies are necessary to determine the key mechanisms of oxytocin receptor resensitization.

The Effect of Extracellular Calcium on Oxytocin-Induced Contractility in Naive and Oxytocin-Pretreated Human Myometrium In Vitro.

BACKGROUND: Prolonged exposure to oxytocin during augmentation of labor is a significant risk factor for uterine atony, resulting in the desensitization phenomenon, a decrease in the responsiveness of myometrium to further oxytocin. The importance of extracellular calcium is well established in spontaneous myometrial contractility; however, its significance is unknown in the context of desensitized myometrium. We aimed to investigate the effect of low, normal, and high extracellular calcium concentration on oxytocin-induced contractility in oxytocin-pretreated human myometrium in vitro. We hypothesized that extracellular normocalcemia would provide superior oxytocin-induced contractility in both naive and oxytocin-pretreated myometrium compared with hypocalcemia and hypercalcemia. METHODS: Myometrial tissue was obtained from women undergoing elective cesarean deliveries and was dissected into longitudinal strips. Each strip was mounted in a single organ bath with physiological salt solution (PSS) under homeostatic conditions and then pretreated for 2 hours with either oxytocin 10 M or PSS (control). The tissue was then washed with PSS, and calcium concentrations were altered to reflect low (1.25 mM), normal (2.5 mM), or high (3.75 mM) levels, thereby providing 6 study groups. After equilibration in the desired calcium concentration, a dose-response testing to oxytocin 10 M to 10 M was performed. Contractile parameters were measured and compared among groups after square root transformation. The primary outcome was motility index (frequency × amplitude), and secondary outcomes included frequency, amplitude, and area under the curve. RESULTS: One hundred seventy-four experiments were conducted from samples obtained from 36 women. In the control group, the mean motility index (√g·contractions/10 min) was significantly lower in the hypocalcemic group than in the normocalcemic group (estimated difference, -0.43; 95% confidence interval [CI], -0.82 to -0.04; P = 0.03). In addition, the mean frequency of contractions (√contractions/10 min) was significantly lower in the hypocalcemic (estimated difference, -0.27; 95% CI, -0.46 to -0.09; P = 0.002) and hypercalcemic groups (estimated difference, -0.18; 95% CI, -0.34 to -0.02; P = 0.03) compared with the normocalcemic group. In the oxytocin-pretreated group, there were no significant differences in the values of any of the contractility parameters of the hypocalcemic or hypercalcemic groups compared with the normocalcemic group (mean motility index [√g·contractions/10 min] estimated difference, 0.10; 95% CI, -0.23 to 0.43; P = 0.74 and -0.39; 95% CI, -1.10 to 0.32; P = 0.39, respectively). CONCLUSIONS: In oxytocin-naive myometrium, normocalcemia provides superior oxytocin-induced contractility compared with hypocalcemic and hypercalcemic conditions. We were unable to draw conclusions regarding oxytocin-pretreated myometrium because of the small sample size relative to the large variability of the data. These observations warrant further investigations in laboratory and clinical settings.

In Vitro Comparative Effect of Carbetocin and Oxytocin in Pregnant Human Myometrium with and without Oxytocin Pretreatment.

BACKGROUND: The purpose of this study was to compare in vitro contractile effects of oxytocin and carbetocin on human term pregnant myometrium with and without oxytocin pretreatment. METHODS: This laboratory investigation was conducted on myometrial samples from women undergoing elective cesarean deliveries. The samples were dissected into four strips and suspended in individual organ bath chambers containing physiologic salt solution. After equilibration, they were pretreated with oxytocin 10 M (experimental group) or physiologic salt solution (control group) for 2 h and then subjected to dose-response testing with increasing concentrations of oxytocin or carbetocin (10 to 10 M). The amplitude, frequency, motility index (amplitude × frequency), and area under the curve of contractions were recorded and analyzed during the equilibration and dose-response periods. Comparisons were made between oxytocin-induced and carbetocin-induced contractions in control and oxytocin-pretreated groups. Motility index was the primary outcome measure. RESULTS: Sixty-three experiments were performed (carbetocin, n = 31; oxytocin, n = 32) on samples from 18 women. The motility index of contractions (√g.contractions/10 min) produced by oxytocin was significantly higher than carbetocin in both control (regression-estimated difference, 0.857; 95% CI, 0.290 to 1.425; P = 0.003) and oxytocin-pretreated (0.813; 0.328 to 1.299; P = 0.001) groups. The motility index was significantly lower in oxytocin-pretreated groups than their respective controls for both oxytocin (-1.040; -1.998 to -0.082; P = 0.03) and carbetocin (-0.996; -1.392 to -0.560; P < 0.001). CONCLUSIONS: In vitro contractions produced by oxytocin are superior to carbetocin in human myometrium with or without oxytocin pretreatment. Oxytocin pretreatment results in attenuation of contractions induced by both oxytocin and carbetocin.

No cardiomyopathy in X-linked myopathy with excessive autophagy.

In X-linked myopathy with excessive autophagy (XMEA) progressive sarcoplasmic accumulation of autolysosomes filled with undegraded debris leads to atrophy and weakness of skeletal muscles. XMEA is caused by compromised acidification of lysosomes resulting from hypofunction of the proton pump vacuolar ATPase (V-ATPase), due to hypomorphic mutations in VMA21, whose protein product assembles V-ATPase. To what extent the cardiac muscle is affected is unknown. Therefore we performed a comprehensive cardiac evaluation in four male XMEA patients, and also examined pathology of one deceased patient's cardiac and skeletal muscle. None of the symptomatic men (aged 25-48 years) had history or symptoms of cardiomyopathy. Resting electrocardiograms and echocardiographies were normal. MRI showed normal left ventricle ejection fraction and myocardial mass. Myocardial late-gadolinium enhancement was not detected. The deceased patient's skeletal but not cardiac muscle showed characteristic accumulation of autophagic vacuoles. In conclusion, in classic XMEA the myocardium is structurally, electrically and clinically spared.

The Contractile Effects of Oxytocin, Ergonovine, and Carboprost and Their Combinations: an In Vitro Study on Human Myometrial Strips.

BACKGROUND: The objective of this study was to compare the in vitro contractile effects of the combination of oxytocin (low dose and high dose) with either ergonovine or carboprost in myometrial strips from women undergoing cesarean delivery (CD), and to study the effect of oxytocin pretreatment on these contractions. We hypothesized that the use of ergonovine or carboprost in combination with oxytocin would improve contractility compared with oxytocin alone. METHODS: Myometrial samples obtained from women undergoing elective CD were pretreated in organ bath chambers with either oxytocin 10 M (experimental) or physiological salt solution (control) for 2 hours. They were then washed and subjected to dose-response testing with oxytocin, ergonovine, or carboprost (10 to 10 M), either alone or in combination with a fixed low-dose (10 M) (LDOx) or high-dose (10 M) (HDOx) oxytocin. The amplitude, frequency, area under the curve, and motility index (amplitude × frequency) of contractions during the dose-response period were analyzed with linear regression models, and compared among the groups. The primary outcome was the motility index across the study groups. RESULTS: One hundred sixty-nine experiments were done in samples obtained from 56 women. The mean square root of the motility index [standard error] (√g·contractions/10 min) of oxytocin was significantly higher in the control (3.40 [0.24]) versus experimental group (2.02 [0.15]) (P < 0.001). When all control groups were compared, the motility index of oxytocin (3.21 [0.25]) was higher than that of ergonovine (2.13 [0.30], P < 0.001 [multiple comparisons adjusted P value, P < 0.001]), carboprost (1.88 [0.10], P < 0.001 [P < 0.001]), ergonovine + LDOx (2.07 [0.15], P < 0.001 [P < 0.001]), and carboprost + LDOx (1.82 [0.15], P < 0.001 [P < 0.001]) and was not different than that of ergonovine + HDOx (3.39 [0.32], P = 0.68 [P = 0.99]) and carboprost + HDOx (2.68 [0.30], P = 0.20 [P = 0.60]). However, in oxytocin-pretreated groups, carboprost + LDOx (motility index: 2.53 [0.08], P = 0.001 [multiple comparisons adjusted P value, P = 0.002]) and ergonovine + HDOx (2.82 [0.15], P < 0.001 [P < 0.001]) exhibited significantly superior contractility response compared with oxytocin alone, while ergonovine + LDOx (2.47 [0.13], P = 0.01 [P = 0.08]) and carboprost + HDOx (2.51 [0.20], P = 0.05 [P = 0.24]) showed higher mean contractility response compared with oxytocin alone but failed to reach statistical significance in adjusted analyses. CONCLUSIONS: The attenuation of oxytocin-induced contractility in oxytocin-pretreated myometrial strips is in keeping with the previously established oxytocin-receptor desensitization phenomenon. Oxytocin is the most effective of the uterotonics tested if the myometrium is not preexposed to oxytocin. However, in the oxytocin-pretreated myometrium, a synergistic response is evident, and the combination of oxytocin with either ergonovine or carboprost produces superior response compared with oxytocin alone. Further in vivo studies in humans are necessary to determine whether these differences identified in vitro are clinically significant.

Late adult-onset of X-linked myopathy with excessive autophagy.

INTRODUCTION: X-linked myopathy with excessive autophagy (XMEA) is characterized by autophagic vacuoles with sarcolemmal features. Mutations in VMA21 result in insufficient lysosome acidification, causing progressive proximal weakness with onset before age 20 years and loss of ambulation by middle age. METHODS: We describe a patient with onset of slowly progressive proximal weakness of the lower limbs after age 50, who maintains ambulation with the assistance of a cane at age 71. RESULTS: Muscle biopsy at age 66 showed complex muscle fiber splitting, internalized capillaries, and vacuolar changes characteristic of autophagic vacuolar myopathy. Vacuoles stained positive for sarcolemmal proteins, LAMP2, and complement C5b-9. Ultrastructural evaluation further revealed basal lamina reduplication and extensive autophagosome extrusion. Sanger sequencing identified a known pathologic splice site mutation in VMA21 (c.164-7T>G). CONCLUSIONS: This case expands the clinical phenotype of XMEA and suggests VMA21 sequencing be considered in evaluating men with LAMP2-positive autophagic vacuolar myopathy.

Progressive myoclonus epilepsy.

The progressive myoclonus epilepsies (PMEs) consist of a group of diseases with myoclonic seizures and progressive neurodegeneration, with onset in childhood and/or adolescence. Lafora disease is a neuronal glycogenosis in which normal glycogen is transformed into starch-like polyglucosans that accumulate in the neuronal somatodendritic compartment. It is caused by defects of two genes of yet unknown function, one encoding a glycogen phosphatase (laforin) and the other an ubiquitin E3 ligase (malin). Early cognitive deterioration, visual seizures affecting over half, and slowing down of EEG basic activity are three major diagnostic clues. Unverricht-Lundborg disease is presently thought to be due to damage to neurons by lysosomal cathepsins and reactive oxygen species due to absence of cystatin B, a small protein that inactivates cathepsins and, by ways yet unknown, quenches damaging redox compounds. Preserved cognition and background EEG activity, action myoclonus early morning and vertex spikes in REM sleep are the diagnostic clues. Sialidosis, with cherry-red spot, neuronopathic Gaucher disease, with paralysis of verticality, and ataxia-PME, with ataxia at onset in the middle of the first decade, are also lysosomal diseases. How the lysosomal defect culminates in myoclonus and epilepsy in these conditions remains unknown.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

The autosomal recessively inherited progressive myoclonus epilepsies and their genes.

Autosomal recessively inherited progressive myoclonus epilepsies (PMEs) include Lafora disease, Unverricht-Lundborg disease, the neuronal ceroid lipofuscinoses, type I sialidosis (cherry-red spot myoclonus), action myoclonus-renal failure syndrome, and type III Gaucher disease. Almost all the autosomal recessively inherited PMEs are lysosomal diseases, with the exception of Lafora disease in which neither the accumulating material nor the gene products are in lysosomes. Progress in identifying the causative defects of PME is near-complete. Much work lies ahead to resolve the pathobiology and neurophysiology of this group of devastating disorders.

VMA21 deficiency causes an autophagic myopathy by compromising V-ATPase activity and lysosomal acidification.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.