Ovarian hyperstimulation syndrome: pathophysiology and prevention

To review and discuss the pathophysiology and prevention strategies for ovarian hyperstimulation syndrome (OHSS), which is a condition that may occur in up to 20% of the high risk women submitted to assisted reproductive technology cycles. The English language literature on these topics were reviewed through PubMed and discussed with emphasis on recent data. The role of estradiol, luteinizing hormone, human chorionic gonadotropin (hCG), inflammatory mediators, the renin-angiotensin system and vascular endothelial growth factor is discussed in the pathophysiology of OHSS. In addition we consider the prevention strategies, including coasting, administration of albumin, renin-angiotensin system blockage, dopamine agonist administration, non-steroidal anti-inflammatory administration, GnRH antagonist protocols, reducing hCG dosage, replacement of hCG and in vitro maturation of oocytes (IVM). Among the many prevention strategies that have been discussed, the current evidence points to the replacement of hCG by GnRH agonists in antagonist cycles and the performance of IVM procedures as the safest approaches.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Aging per se does not influence glucose homeostasis - In vivo and in vitro evidence

OBJECTIVE - To assess the effect of age on glucose metabolism by examining 1) glucose metabolism in young and middle-aged subjects when total or regional adiposity is taken into account and 2) in vitro glucose transport in adipose tissue explants from young and middle-aged women paired for total and abdominal adiposity. RESEARCH DESIGN AND METHODS - Study 1: body composition, subcutaneous abdominal and visceral adipose tissue areas, and fasting and oral glucose-stimulated glucose and insulin were measured in 84 young and 81 middle-aged men and in 110 young and 91 middle-aged women. Study 2: glucose uptake in subcutaneous abdominal and visceral adipose tissue explants were measured in eight young and eight middle-aged women. RESULTS - Study 1: young and middle-aged men showed similar subcutaneous abdominal tissue area, whereas fat mass and visceral adipose tissue were greater in middle-aged than in young men (P < 0.01). Fat mass and subcutaneous and visceral adipose tissue areas were greater in middle-aged as compared with young women (P < 0.01). Fasting plasma glucose and the glucose response to an oral glucose tolerance test were significantly higher in middle-aged than in young men and women (P < 0.001). Statistical control for visceral adipose tissue area eliminated the difference seen in glucose response in men and women. Study 2: glucose transport in subcutaneous and omental adipose tissue did not differ between young and middle-aged women. CONCLUSIONS - 1) Visceral obesity, more than age per se, correlates with glucose intolerance in middle-aged subjects; 2) aging does not influence in vitro adipose tissue glucose uptake.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

In Vitro Methods for Specific IgE Detection on Cow’s Milk Allergy

Background: A new method for determining serum specific IgE (IMMULITE“ 2000 3gAllergy) has recently become available. Objective: To evaluate the clinical performance of IMMULITE 2000 in the diagnosis of cow’s milk allergy compared with that of UniCAP“. Additionally, we verified the behavior of both methods at two diagnostic decision points proposed by other authors. Methods: The study population consisted of 31 children with cow’s milk allergy (group A) and a control group of 19 atopic children without food allergy(group B). A blood sample from each child was tested using both methods and the results were compared. Results: In group A, the values for cow’s milk IgE ranged from 0.35 kU/L (the lowest common detection limit) to above 100 kU/L. In group B, the values were less than 1.1 kU/L for IMMULITE 2000 and less than 1.6 kU/L for UniCAP. An agreement of 90 % in IgE classes was obtained. Both methods demonstrated exactly the same diagnostic performance(sensitivity: 100 %; specificity: 78.9 %; negative predictive value: 100%; positive predictive value: 84.6%;efficiency: 90.2 %). The evaluation of the two methods at the two different decision points proposed in the literature showed a better positive predictive value with UniCAP, but we obtained equivalent performance with IMMULITE 2000 by choosing higher cutoff values. Conclusions: We conclude that IMMULITE 2000 is as effective as UniCAP in the diagnosis of cow’s milk allergy. Both methods can be used to obtain site-specific decision points that are population, age and disease dependent.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS: Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

O efeito da insulina na placentação: estudos in vitro com células HTR8-SVneo

Dissertação de mestrado em Bioquímica Aplicada

Couple's willingness to donate embryos for research: a longitudinal study

Introduction. Decision-making on embryo disposition is a source of distress and is subject to change over time. This paper analyses the willingness of couples undergoing in vitro fertilization to donate cryopreserved embryos for research from 15 days after embryo transfer to 12 months later, taking into account the influence of psychosocial, demographic, and reproductive factors. Materials and methods. Prospective longitudinal study, with 74 heterosexual couples undergoing in vitro fertilization in a public fertility centre in Portugal, recruited between 2011 and 2012. Participants were evaluated twice: 15 days after embryo transfer and 12 months later. Results. A significant decrease in patients’ willingness to donate embryos for research over time was observed [86.5% to 73.6%; relative risk (RR) = 0.85; 95% CI 0.76–0.95]. A higher education level (>12 years) [adjusted RR (RRadj) = 0.79; 95% CI 0.64–0.96], considering research on human embryos to be important (vs. very important) (RRadj = 0.59; 95% CI 0.39–0.85) and practicing a religion less than once a month (vs. at least once a month) (RRadj = 0.73; 95% CI 0.53–1.00) seemed associated with unwillingness to donate embryos for research over time. Change towards non-donation happened mainly among couples who first considered that it was better to donate than wasting the embryos. Change towards donation occurred mostly among those stating that their priority at time 1 was to have a baby and who became pregnant in the meantime. Conclusions. Quality of care guided by patients’ characteristics, values, preferences, and needs calls for considering the factors and reasons underlying couples’ willingness to donate embryos for research over time as a topic in psychosocial guidelines for infertility and medically assisted reproductive care.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro.

There is a clinical need to enhance functional recovery of injured peripheral nerves. Local administration of neurotrophic factors (NTFs) after surgical repair has been proposed for this purpose. Little is known, however, on the optimal local dose and dosing frequency of NTFs in a peripheral nerve defect. For increasing our knowledge on biologically relevant local NTFs concentrations and for making available an in vitro assay for assessing the bioactivity of NTFs in connection with implantable localized delivery systems, we developed in this study a bioassay for NTFs, which is based on dorsal root ganglion (DRG) explants from E9 (9 days old) chicken embryos. Axonal elongation and extent of axonal branching was analyzed microscopically after addition of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), each alone and in combination. GDNF significantly promoted axonal elongation, but only little axonal branching, whereas NGF induced extensive axonal branching with modest axonal elongation. The combination of GDNF and NGF exerted a synergistic effect on the axonal elongation, axonal branching and growth kinetics. GDNF and NGF also enhanced the expression of their respective functional receptors Ret and TrkA on the DRG neurons. This information should be relevant for the development of implants containing NTFs and on drug therapy of damaged peripheral nerves.

Avaluació de la teratogènia in vitro mitjançant el cultiu d’embrions sencers (WEC)

Projecte de recerca elaborat a partir d’una estada a la Charité - Universitätsmedizin Berlin, Alemanya, entre novembre i desembre del 2007. En aquest treball es presenta el protocol a seguir per a dur a terme el cultiu d’embrions sencers in vitro (Whole Embryo Culture, WEC). Amb aquest protocol es pretén implementar la tècnica del WEC en el laboratori de la Unitat de Toxicologia de la Facultat de Farmàca (UB), seguint la metodologia apresa durant l’estada i deixant per escrit tots els passos seguits i el material i la metodologia concreta de cadascun d’ells. En el WEC es cultiven embrions de rata de 9.5 dies durant 48h en ampolles rotatòries en un medi líquid i amb una fase gasosa controlats. Durant el cultiu, tenen lloc dos processos principals: el plegament de l’embrió i l’organogènesi. Els embrions durant els dos dies que dura el cultiu es pleguen en els plans transversal i sagital, passant d’un embrió pla a un altre de cilíndric en forma de “C”. En aquest període, a més, es produeixen importants processos d’organogènesi com la neurulació, la formació de la cresta neural, dels somites, dels vasos sanguinis - el cor inclòs- i de la sang. Es comencen a formar la placoda nasal, la vesícula oftàlmica, la vesícula òtica, les extremitats superiors i inferiors i la cua. En la memòria adjunta es descriuen amb detall els processos d'aparellament dels animals, preparació del material i del medi de cultiu, el procés d'aïllament del embrions en el dia 9.5, les condicions de cultiu i l'avaluació dels embrions en el dia 11.5. Finalment es presenten resultats d'embrions en situació control amb un correcte desenvolupament i es mostra com, al final de l'estada, es va aconseguir el cultiu d’embrions control amb un desenvolupament correcte i estadísticament sense diferències respecte als diferents paràmetres mesurats en comparació amb els embrions control de la Charité-Universitätsmedizin de Berlin.

Arrhythmogenesis in the developing heart during anoxia-reoxygenation and hypothermia-rewarming: an in vitro model.

INTRODUCTION: The spatio-temporal pattern of arrhythmias in the embryonic/fetal heart subjected to a transient hypoxic or hypothermic stress remains to be established. METHODS AND RESULTS: Spontaneously beating hearts or isolated atria, ventricles, and conotruncus from 4-day-old chick embryos were subjected in vitro to 30-minute anoxia and 60-minute reoxygenation. Hearts were also submitted to 30-minute hypothermia (0-4 degrees C) and 60-minute rewarming. ECG disturbances and alterations of atrial and ventricular electromechanical delay (EMD) were systematically investigated. Baseline functional parameters were stable during at least 2 hours. Anoxia induced tachycardia, followed by bradycardia, atrial ectopy, first-, second-, and third-degree atrio-ventricular blocks and, finally, transient electromechanical arrest after 6.8 minutes, interquartile ranges (IQR) 3.1-16.2 (n = 8). Reoxygenation triggered also Wenckebach phenomenon and ventricular escape beats. At the onset of reoxygenation QT, PR, and ventricular EMD increased by 68%, 70%, and 250%, respectively, whereas atrial EMD was not altered. No fibrillations, no ventricular ectopic beats, and no electromechanical dissociation were observed. Arrhythmic activity of the isolated atria persisted throughout anoxia and upon reoxygenation, whereas activity of the isolated ventricles abruptly ceased after 5 minutes of anoxia and resumed after 5 minutes of reoxygenation. During hypothermia-rewarming, cardiac activity stopped at 17.9 degrees C, IQR 16.2-20.6 (n = 4) and resumed at the same temperature with no arrhythmias. All preparations fully recovered after 40 minutes of reoxygenation or rewarming. CONCLUSION: In the embryonic heart, arrhythmias mainly originated in the sinoatrial tissue and resembled those observed in the adult heart. Furthermore, oxygen readmission was by far more arrhythmogenic than rewarming and the chronotropic, dromotropic, and inotropic effects were fully reversible.

The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro.

TMPRSS3 encodes a transmembrane serine protease that contains both LDLRA and SRCR domains and is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). To study its function, we cloned the mouse ortholog which maps to Mmu17, which is structurally similar to the human gene and encodes a polypeptide with 88% identity to the human protein. RT-PCR and RNA in situ hybridization on rat and mouse cochlea revealed that Tmprss3 is expressed in the spiral ganglion, the cells supporting the organ of Corti and the stria vascularis. RT-PCR on mouse tissues showed expression in the thymus, stomach, testis and E19 embryos. Transient expression of wild-type or tagged TMPRSS3 protein showed a primary localization in the endoplasmic reticulum. The epithelial amiloride-sensitive sodium channel (ENaC), which is expressed in many sodium-reabsorbing tissues including the inner ear and is regulated by membrane-bound channel activating serine proteases (CAPs), is a potential substrate of TMPRSS3. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. In contrast, 6 TMPRSS3 mutants (D103G, R109W, C194F, W251C, P404L, C407R) causing deafness and a mutant in the catalytic triad of TMPRSS3 (S401A), failed to undergo proteolytic cleavage and activate ENaC. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggest: (i) the existence of an auto-catalytic processing by which TMPRSS3 would become active, and (ii) that ENaC could be a substrate of TMPRSS3 in the inner ear.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.