Ovarian response prediction in GnRH antagonist treatment for IVF using anti-Müllerian hormone.

STUDY QUESTION: What is the clinical value of anti-Müllerian hormone (AMH) for the prediction of high or low ovarian response in controlled ovarian stimulation for IVF using GnRH antagonist treatment? SUMMARY ANSWER: AMH as a single test has substantial accuracy for ovarian response prediction in GnRH antagonist treatment for IVF, with a higher accuracy for predicting a high response than for low response. WHAT IS KNOWN ALREADY: The role of AMH and other patient characteristics in ovarian response prediction has been studied extensively in long GnRH agonist protocols; however, little information is available regarding the clinical value in GnRH antagonists. STUDY DESIGN, SIZE, DURATION: This is an observational (retrospective) substudy as part of an ongoing cohort study. A total of 487 patients scheduled for IVF/ICSI between 2006 and 2011 were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a regular cycle who underwent their first IVF/ICSI cycle with GnRH antagonist treatment while receiving a starting dose of 150 or 225 IU recombinant FSH were included in the study. Patients were divided into three subgroups according to the following ovarian response categories: high (>15 oocytes or cycle cancellation), normal (4-15 oocytes) and low (<4 oocytes or cycle cancellation). Serum samples collected prior to IVF treatment were used to determine serum AMH levels. MAIN RESULTS AND THE ROLE OF CHANCE: According to the predefined ovarian response categories, 58 patients were classified as high, 326 as normal and 101 as low responders, and the ongoing pregnancy rates did not differ among groups (19.0, 22.1 and 16.8%, respectively, P = 0.9). For the prediction of high response, AMH had an area under the receiver-operating characteristic curve (AUC) of 0.87. Both female age and BMI had lower accuracy (AUC 0.66 and 0.58, respectively). For low response prediction, again AMH had a better accuracy (AUC 0.79) than female age and BMI (AUC 0.59 and 0.56, respectively). In a multivariate model, including the factors age, AMH, BMI, smoking, type and duration of subfertility, only BMI added some predictive value to AMH for both high and low response prediction. Clinical test characteristics demonstrated that using a specificity of ∼90%, the detection rate of AMH for high and low response, corresponding with a test cut-off of 4.5 and 0.8 µg/l, was ∼60 and ∼45%, respectively. LIMITATIONS, REASONS FOR CAUTION: The impact of the antral follicle count (AFC) on ovarian response prediction in GnRH antagonists was not assessed; however, previously studies demonstrated that for GnRH antagonists, AMH has a better accuracy than AFC. WIDER IMPLICATIONS OF THE FINDINGS: The current study demonstrates that AMH is an adequate predictor for both high and low response in GnRH antagonist cycles, showing a similar accuracy to GnRH agonists, as reported previously. The optimization and individualization of GnRH antagonist protocols may be improved by using an AMH-tailored approach. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Academic Institutional Resources of the Department of Reproductive Medicine of the UMC Utrecht. O.H., M.J.C.E, E.W.G.L and H.L.T. have nothing to declare. N.S.M. has received fees and/or grant support from the following companies (in alphabetic order): Anecova, Ferring, Informa, Merck Serono and MSD. B.C.J.M.F. has received fees and/or grant support from the following companies (in alphabetic order); Childhealth, CVON, Ferring, Ova-Science, PregLem, Roche and Watson laboratories. F.J.B. has received fees and/or grant support from the following companies (in alphabetic order); Merck Serono and MSD. TRIAL REGISTRATION NUMBER: www.clinicaltrials.gov, Protocol ID 13-109.

Column chromatographic characterization of complex formation of pro-IGF-II isoforms with acid labile subunit and IGF-binding proteins associated with non-islet cell tumour induced hypoglycaemia.

OBJECTIVE AND DESIGN: Non-islet cell tumour induced hypoglycaemia (NICTH) is a paraneoplastic phenomenon that is associated with the formation of several isoforms of pro-insulin like growth factor 2 (pro-IGF-II), or so called "big" IGF-II. Disturbance of ternary complex formation by big IGF-II is assumed to be a crucial early event in the pathogenic cascade of hypoglycaemia. By size-exclusion chromatography, we investigated complex formation by adding different naturally occurring isoforms of pro-IGF-II to pooled normal adult serum. Results were compared with the analysis of the serum from a patient with NICTH. RESULTS: Gel filtration experiments with the serum of a patient with NICTH demonstrated that ternary complex formation was severely compromised. The various forms of pro-IGF-II did not induce a shift of IGF-binding protein 3 (IGFBP-3) from 150kD towards smaller binary complexes in the normal adult serum, suggesting that they did not interfere with the interaction between the acid labile subunit and IGFBP-3. Instead, unglycosylated recombinant pro-IGF-II[1-104] was capable of forming a 150kD complex. In contrast, predominantly glycosylated and unglycosylated pro-IGF-II[1-87] eluted in the free unbound form. We showed that mature IGF-II and isoforms of pro-IGF-II were able to phosphorylate the IGF-I receptors of MC7 cells, albeit to a markedly lesser extent than IGF-I. When the patient's serum was tested in this system, the IGF-I receptor phosphorylation activity was considerably less than that in sera from age matched healthy individuals. CONCLUSION: We postulate that, alongside the presence of big IGF-II in the circulation, additional steps are required to stimulate the release of IGF-II and pro-IGF-II isoforms from IGFBPs in vivo. These factors may be proteases, that are present in the local environment of the tumour and in insulin-sensitive tissues.

Genome-wide association study of monoamine metabolite levels in human cerebrospinal fluid.

Studying genetic determinants of intermediate phenotypes is a powerful tool to increase our understanding of genotype-phenotype correlations. Metabolic traits pertinent to the central nervous system (CNS) constitute a potentially informative target for genetic studies of intermediate phenotypes as their genetic underpinnings may elucidate etiological mechanisms. We therefore conducted a genome-wide association study (GWAS) of monoamine metabolite (MM) levels in cerebrospinal fluid (CSF) of 414 human subjects from the general population. In a linear model correcting for covariates, we identified one locus associated with MMs at a genome-wide significant level (standardized ß=0.32, P=4.92 × 10(-8)), located 20 kb from SSTR1, a gene involved with brain signal transduction and glutamate receptor signaling. By subsequent whole-genome expression quantitative trait locus (eQTL) analysis, we provide evidence that this variant controls expression of PDE9A (ß=0.21; P unadjusted=5.6 × 10(-7); P corrected=0.014), a gene previously implicated in monoaminergic transmission, major depressive disorder and antidepressant response. A post hoc analysis of loci significantly associated with psychiatric disorders suggested that genetic variation at CSMD1, a schizophrenia susceptibility locus, plays a role in the ratio between dopamine and serotonin metabolites in CSF. The presented DNA and mRNA analyses yielded genome-wide and suggestive associations in biologically plausible genes, two of which encode proteins involved with glutamate receptor functionality. These findings will hopefully contribute to an exploration of the functional impact of the highlighted genes on monoaminergic transmission and neuropsychiatric phenotypes.

Hereditary persistence of alpha-fetoprotein (HPAF P): review of the literature.

Alpha-fetoprotein (AFP) serum levels are raised in several clinical conditions, ranging from non-pathological conditions to malignancies. Hereditary persistence of alpha-fetoprotein (HPAFP) is a rare benign disorder with elevated AFP levels. HPAFP is described as a benign autosomal dominantly inherited condition which is not associated with any clinical disability or additional symptoms. In the past 28 years, only 19 families have been described; due to this unfamiliarity with HPAFP, elevated AFP levels are never attributed to HPAFP. However, undiagnosed HPAFP can result in inappropriate and unnecessary treatment decisions. Therefore, HPAFP should be taken into consideration in patients with unexplained elevated AFP levels, and especially in patients with urological disorders.

Heterophile antibodies rarely influence the measurement of thyroglobulin and thyroglobulin antibodies in differentiated thyroid cancer patients.

The aim of the study was to determine the impact of heterophile antibodies on the measurement of serum thyroglobulin (Tg), thyroglobulin recovery, and thyroglobulin antibody levels in differentiated thyroid carcinoma patients. We studied serum samples of 201 individual patients that were followed in our hospital for differentiated thyroid carcinoma and 52 control samples. Samples were split; half were treated by incubating the sample for 1 h in HAB-blocking tubes, the remainder was left untreated. Subsequently thyroglobulin and thyroglobulin antibody levels were measured in both the blocked and untreated samples. A difference between the two samples was considered significant if the blocked sample deviated from the untreated one by more than 2.77 times the standard deviation for the method. In the measurement of Tg, 2 patients showed a moderate, but significant lowering of Tg levels after blocking treatment, but not so great as to affect clinical management. None of the 52 controls showed heterophile antibody interference in thyroglobulin measurement. Neither in DTC patients, nor in controls was any possible heterophile antibody interference encountered. And in all thyroid carcinoma patients, and in all but one controls, no interference was found in the thyroglobulin antibody measurement. All in all a possible heterophile antibody interference was found in 3/759 tests (0.4%). We can assume that heterophile antibody interference is not a factor to be reckoned with in the daily practice of Tg measurement in the treatment and follow-up of differentiated thyroid carcinoma patients.

Value of diagnostic radioiodine scintigraphy and thyroglobulin measurements after rhTSH injection.

UNLABELLED: Measurements of thyroglobulin (Tg) levels 72 h after administration of recombinant human thyrotropin (rhTSH) are recommended by the manufacturer in the follow-up of patients with differentiated thyroid carcinoma (DTC). In our department, Tg measurements are performed both 24 h and 72 h after administration of rhTSH, together with 72 h post rhTSH 131I whole body scintigraphy (WBS). The OBJECTIVE of this study is to compare the diagnostic usefulness of Tg measurements 24 and 72 h after rhTSH administration, and 131I WBS. PATIENTS AND METHODS: 181 patients were included who had been referred to our Nuclear Medicine Department for follow-up after 131I ablation of DTC. Tg measurements 24 h (Tg24) and 72 h (Tg72) after rhTSH, and 131I WBS, were done in all patients. The lower detection limit of Tg was 0,2 microg/l. RESULTS: 47 patients (26%) had detectable Tg levels: in 4/47 cases (8%) only Tg24 was detectable (always <1 microg/l), and in 6/47 cases (11%), only Tg72 was detectable. In 10/47 patients with detectable Tg-levels, Tg24 and Tg72 tested equally. In 27/47 cases, Tg24 was lower, and in 10/47 higher, than Tg72. Two patients with one or two positive Tg-test results also had a positive 131I WBS. In 8 patients (14%) only the 131I WBS was positive; an anatomical substrate for such a Tg-negative positive WBS was confirmed in only 2 patients. CONCLUSION: Tg-measurement 72 hours after rhTSH injection reveals all clinically relevant detectable Tg-levels. Diagnostic 131I scintigraphy may be omitted, even in high-risk patients.

Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF.

BACKGROUND: The study of human endometrial-embryonic interactions is complicated by the disruptive impact of endometrial sample collection on the process of implantation itself. Endometrial secretion analysis is a novel technique, non-disruptive to implantation. The primary aim of this prospective cohort study was to explore whether a cytokine profile predictive of implantation and clinical pregnancy can be identified in endometrial secretions aspirated immediately prior to embryo transfer following IVF. METHODS: Endometrial secretions, aspirated immediately prior to embryo transfer from 210 women undergoing IVF, were analyzed using a multiplex immunoassay for 17 soluble regulators of implantation, namely interleukin (IL)-1beta, IL-5, IL-6, IL-10, IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, macrophage migration inhibitory factor, eotaxin, IFN-gamma-inducible 10 kDa protein (IP-10), monocyte chemo-attractant protein-1 (MCP-1), Dickkopf homolog 1, heparin-binding epidermal growth factor and vascular endothelial growth factor (VEGF). In order to detect implantation, daily urine samples were collected after embryo transfer, and human Chorionic Gonadotropin (hCG) concentrations were analyzed by an immunoassay. RESULTS: Multivariable logistic regression analysis revealed significant associations (negative and positive association, respectively) between MCP-1 (P = 0.005) and IP-10 (P = 0.037) levels and implantation, and between IL-1beta (P = 0.047) and TNF-alpha (P = 0.023) levels and clinical pregnancy. The predictive value for pregnancy of IL-1beta and TNF-alpha was observed to be equivalent and additive to that of embryo quality. CONCLUSIONS: Endometrial secretion cytokine profiling offers a novel, non-disruptive approach to study the role of the endometrium in human embryo implantation and identifies a profile which appears to be conducive to clinical pregnancy. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov (nCT00264992).

Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study.

Cisplatin-induced toxicities are mainly caused by the formation of free radicals, leading to oxidative organ damage. Plasma concentrations of antioxidants decrease significantly during cisplatin chemotherapy for cancer. Forty-eight cancer patients treated with cisplatin-based chemotherapy were randomised in a double-blind manner to receive either supplementation with vitamin C, vitamin E and selenium dissolved in a beverage or to receive a placebo beverage. Primary outcome measures were the amount of nephrotoxicity and ototoxicity induced by cisplatin. No significant differences were found between the two study groups with respect to these primary outcome measures. However, patients who achieved the highest plasma concentrations of the three antioxidant micronutrients had significantly less loss of high-tone hearing. In addition, significant correlations were found between the reduced/oxidised vitamin C ratio and malondialdehyde (MDA), markers of oxidative stress, and cisplatin-induced ototoxicity and nephrotoxicity. The lack of protection against cisplatin-induced toxicities in patients in the intervention arm may be related to poor compliance and/or inadequate supplementation. Supplementation with a higher dose (intensity) and in combination with other antioxidants should be investigated further.

Detection of circulating Tg-mRNA in the follow-up of papillary and follicular thyroid cancer: how useful is it?

To investigate the usefulness of thyroglobulin mRNA (Tg-mRNA) detection in peripheral blood in the follow-up of papillary and follicular (differentiated) thyroid cancer, a literature study was performed. Both evidence for and evidence against the usefulness of Tg-mRNA detection were found. Also, evidence for the expression of Tg-mRNA in cells other than normal or neoplastic thyroid follicular cells was found. It is concluded that currently Tg-mRNA detection is not a useful tool in the follow-up of differentiated thyroid carcinoma, but that the concept of using RT-PCR measurements during follow-up still warrants further research.

Non-protein bound iron release during chemotherapy in cancer patients.

Non-protein bound iron (NPBI) is able to catalyse oxidative reactions, causing damage to vital structures. Adverse effects induced by cisplatin seem, in part, to be mediated by free radicals. In the present study, we have measured plasma NPBI, various other iron parameters and antioxidants in 28 cancer patients undergoing cisplatin-based chemotherapy at various time points before and during chemotherapy. No NPBI was present prior to therapy, but within 1-4 days following the first administration of chemotherapy, mean NPBI rose significantly to 10.6+/-6.6 micromol/l (range, 0.6-21.3 micromol/l) in 18 (64.3%) of the 28 patients measured. The rise in NPBI was accompanied by a significant rise in total plasma iron and ferritin and a marked decrease in the latent iron-binding capacity. Concomitantly, plasma vitamins C and E decreased significantly, indicating consumption of antioxidants. Similar observations were also made during the fourth chemotherapy cycle. The increase in NPBI preceded and correlated significantly with chemotherapy toxicity, such as a decrease in leucocyte count and haemoglobin, with a transient rise in various liver enzymes and with known cisplatin-related toxicity, i.e. the loss of renal and hearing function. In conclusion, cisplatin chemotherapy induces oxidative damage which rapidly leads to release of iron from intracellular proteins and the appearance of NPBI. Bone marrow, red blood cells, liver and kidney seem to be a likely source of NPBI. The observed high levels of NPBI may be a major causative determinant in chemotherapy-induced toxicity.

A potential role for the mast cell in the pathogenesis of idiopathic osteoporosis in men.

Osteoporosis is increasingly being recognized in men. Secondary causes are often implicated, but the mechanism of bone loss remains unclear in about a third of patients. The mast cell is a complex cell that stores a number of factors known to affect bone metabolism. Patients with systemic mastocytosis often demonstrate osteoporosis and bone marrow mast cells may be increased in osteoporotic postmenopausal women. We address the possible role of the mast cell in the pathophysiology of male osteoporosis by studying the relationship between bone marrow infiltration with mast cells and the 24 h urine excretion of N-methylhistamine, and the severity of osteoporosis in 48 consecutive men with idiopathic osteoporosis (bone mineral density Z score of <-1 and/or at least one prevalent vertebral fracture). Secondary causes for osteoporosis were excluded and none of the patients had systemic manifestations of enhanced mast cell activity. A widely variable number of morphologically normal mast cells were counted in toluidine blue-stained sections from 42 of 46 evaluable bone marrow biopsies. In 4 of the 42 biopsies (9%), clusters of abnormal mast cells were identified. These four patients were the only ones who also demonstrated increased 24 h urine excretion of N-methylhistamine. There was a significant positive relationship between mast cell number and the 24 h urine excretion of N-methylhistamine reflecting mast cell activity (p = 0.0001), and this latter measurement correlated negatively with bone mineral density (BMD) at the lumbar spine (p < 0.001). We identified clinically important bone marrow cell infiltration with pathologic mast cells in the absence of systemic manifestations of mast cell hyperactivity as an additional secondary cause for osteoporosis in some 9% of men with idiopathic osteoporosis, and found urinary excretion of N-methylhistamine to be above the upper limit of the normal laboratory reference range diagnostic for this cause of secondary osteoporosis. The more continuous spectrum in the relationship between mast cell activity and BMD supports a potential role for this cell in the pathogenesis of idiopathic male osteoporosis.

LPS-induced IL-10 production in whole blood cultures from chronic fatigue syndrome patients is increased but supersensitive to inhibition by dexamethasone.

Several causes have been held responsible for the chronic fatigue syndrome (CFS), including an altered hypothalamus-pituitary-adrenal gland (HPA)-axis activity, viral infections and a reduced Th1 activity. Therefore, it was investigated whether the regulation of IL-10 is different in CFS. LPS-induced cytokine secretion in whole blood cultures showed a significant increase in IL-10 and a trend towards a decrease in IL-12 as compared with healthy controls. In patients and controls, IL-12 secretion was equally sensitive to suppression by dexamethasone, whereas IL-10 secretion appeared more sensitive in CFS-patients. In controls, IL-10 and IL-12 secretion were inversely correlated with free serum cortisol (r=-0.492, p<0.02 and r=-0.434, p<0.05, respectively). In CFS, such an inverse correlation was found for IL-12 (r=-0.611, p<0.02) but not for IL-10 (r=-0.341, ns). These data are suggestive for a disturbed glucocorticoid regulation of IL-10 in CFS.

Increased sensitivity to glucocorticoids in peripheral blood mononuclear cells of chronic fatigue syndrome patients, without evidence for altered density or affinity of glucocorticoid receptors.

BACKGROUND: In this study we tested the hypothesis that the increased sensitivity to glucocorticoids in chronic fatigue syndrome (CFS)-patients can be attributed to an altered functioning of their glucocorticoid receptors (GR). METHODS: For this purpose, affinity and distribution of the GR were studied in purified, peripheral blood mononuclear cells (PBMC) of 10 CFS patients and 14 controls along with the responsiveness of these cells to glucocorticoids in vitro. RESULTS: Affinity (Kd) and number of GR was not different in PBMC of CFS patients when compared with the controls (Kd, 12.9 +/- 8.9 nmol vs 18.8 +/- 16.2 nmol and GR number, 4,839 +/- 2,824/ cell vs 4,906 +/- 1,646/cell). Moreover, RT-PCR revealed no differences in GR messenger RNA expression. Nevertheless, PBMC from CFS patients showed an increased sensitivity to glucocorticoids in vitro. In CFS patients 0.01 micromol dexamethasone suppressed PBMC proliferation by 37%, whereas the controls were only suppressed by 17% (P < 0.01). Addition of phorbol 12-myristate 13-acetate to the cultures rendered the cells resistant to dexamethasone with regard to proliferation and IL-10 and IFN-gamma production, but not to IL-2 and TNF-alpha production in both patients and controls. No difference between patients and controls was observed in this respect CONCLUSIONS: In conclusion, PBMC of CFS patients display an increased sensitivity to glucocorticoids, which cannot be explained by number or affinity of the GR but should rather be attributed to molecular processes beyond the actual binding of the ligand to the GR.

Dexamethasone concentration in the subretinal fluid after a subconjunctival injection, a peribulbar injection, or an oral dose.

PURPOSE: To determine dexamethasone concentrations in the subretinal fluid of patients after a peribulbar injection, a subconjunctival injection, or an oral dose of dexamethasone and to compare the results with those of previous similar studies of dexamethasone concentrations in the vitreous. DESIGN: Prospective, nonrandomized, comparative trial. PARTICIPANTS: One hundred forty-eight patients with a rhegmatogenous retinal detachment. METHODS: Fifty patients received a peribulbar injection of 5 mg dexamethasone disodium phosphate, 49 received a subconjunctival injection of 2.5 mg dexamethasone disodium phosphate, and 49 received an oral dose of 7. 5 mg dexamethasone at various time intervals before surgery. At the time of surgery, a subretinal fluid sample was taken from each patient. MAIN OUTCOME MEASURES: The dexamethasone concentration in the subretinal fluid measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentrations in the subretinal fluid after the peribulbar injection, the subconjunctival injection, and the oral dose were, respectively, 82.2 ng/ml (standard error, 17. 6), 359 ng/ml (standard error, 80.2), and 12.3 ng/ml (standard error, 1.61). Corrected for dose, the maximum dexamethasone concentrations after subconjunctival injection and peribulbar injection were, respectively, 120 (95% confidence interval, 54/180) and 13 (95% confidence interval, 6.8/20) times greater than after oral administration. CONCLUSIONS: A subconjunctival injection of dexamethasone disodium phosphate is more effective in delivering dexamethasone into the subretinal fluid of patients with a rhegmatogenous retinal detachment compared with peribulbar injection or oral administration. The subretinal dexamethasone concentrations were higher than concentrations measured in the vitreous in previous studies with a similar setup after all three delivery methods.

Aprotinin administration in the pericardial cavity does not prevent platelet activation.

OBJECTIVES: Aprotinin is frequently administered systemically to patients undergoing cardiopulmonary bypass to inhibit activation of platelets and plasma protein systems and thus reduce postoperative blood loss. Two reports on local aprotinin administration, that is, into the pericardial cavity, also indicated improvement in postoperative blood loss, but the underlying mechanism was not investigated. We previously reported the disappearance of glycoprotein Ib from the platelet surface and the appearance of platelet-derived microparticles in the pericardial cavity of patients undergoing cardiopulmonary bypass as signs of platelet activation. Here, we investigated whether such local aprotinin administration reduced platelet activation. METHODS: In a double-blind study, 6 patients received aprotinin (500,000 KIU) into the pericardial cavity during the operation and 7 patients received a placebo. Platelet surface glycoprotein Ib expression, concentration of microparticles, and concentration of complexes of platelets with leukocytes, erythrocytes, or each other, were measured by flow cytometry. RESULTS: We confirmed the reduced glycoprotein Ib expression and the increased concentration of microparticles in the pericardial cavity, as previously reported, and found no increased concentration of platelet complexes. However, no differences between aprotinin and placebo treatments were observed in these platelet activation parameters in the pericardial cavity or the systemic circulation. CONCLUSION: We conclude that administration of aprotinin into the pericardial cavity during cardiopulmonary bypass and at concentrations similar to the systemic application does not reduce platelet activation in that compartment or the systemic circulation.

High concentration of dexamethasone in aqueous and vitreous after subconjunctival injection.

PURPOSE: To determine the dexamethasone concentration in aqueous, vitreous, and serum of patients after a subconjunctival injection with dexamethasone disodium phosphate and to compare the effectiveness of a subconjunctival injection as a method of delivering dexamethasone into the vitreous with that of two previously tested routes: peribulbar injection and oral administration. METHODS: In a prospective study, 50 phakic patients who underwent a pars plana vitrectomy received a single subconjunctival injection with 2.5 mg of dexamethasone disodium phosphate, aqueous solution (after topical anesthesia and a subconjunctival injection with lidocaine) at varied intervals before surgery. An aqueous and a vitreous sample were taken from each patient, and serum samples were collected at multiple time points from nine of 50 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentration in the aqueous was 858 ng per ml at 2.5 hours after injection, and in the vitreous, 72.5 ng per ml at 3 hours. In serum, a mean maximum concentration of 32.4 ng per ml was measured at approximately 30 minutes after injection. CONCLUSIONS: Subconjunctival injection of 2.5 mg of dexamethasone disodium phosphate resulted in an estimated vitreous dexamethasone peak concentration three and 12 times higher, respectively, than after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and an oral dose of 7.5 mg of dexamethasone. Thus, a subconjunctival injection is the most effective method of delivering dexamethasone into both the anterior and posterior segments of the eye. Systemic drug absorption is considerable and is of the same order of magnitude as after peribulbar injection.

Variations in melanin formation by cultured melanocytes from different skin types.

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Function of the hypothalamic-pituitary-adrenal axis in patients with fibromyalgia and low back pain.

OBJECTIVE: We suggested fibromyalgia (FM) is a disorder associated with an altered functioning of the stress-response system. This was concluded from hyperreactive pituitary adrenocorticotropic hormone (ACTH) release in response to corticotropin-releasing hormone (CRH) and to insulin induced hypoglycemia in patients with FM. In this study, we tested the validity and specificity of this observation compared to another painful condition, low back pain. METHODS: We recruited 40 patients with primary FM (F:M 36:4), 28 patients (25:3) with chronic noninflammatory low back pain (LBP), and 14 (12:2) healthy, sedentary controls. A standard 100 microg CRH challenge test was performed with measurement of ACTH and cortisol levels at 9 time points. They were also subjected to an overnight dexamethasone suppression test, followed by injection of synthetic ACTH1-24. At 9 AM, the patients divided in 2 groups, received either 0.025 or 0.100 microg ACTH/kg body weight to test for adrenocortical sensitivity. Basal adrenocortical function was assessed mainly by measurement of 24 h urinary excretion of free cortisol. RESULTS: Compared to the controls, the patients with FM displayed a hyperreactive ACTH release in response to CRH challenge (ANOVA interaction effect p = 0.001). The mean ACTH response of the patients with low back pain appeared enhanced also, but to a significantly lesser extent (p = 0.02 at maximum level) than observed in the patients with FM. The cortisol response was the same in the 3 groups. Following dexamethasone intake there were 2 and 4 nonsuppressors in the FM and LBP groups, respectively. The very low and low dose of exogenous ACTH1-24 evoked a dose and time dependent cortisol response, which, however, was not significantly different between the 3 groups. The 24 h urinary free cortisol levels were significantly lower (p = 0.02) than controls in both patient groups; patients with FM also displayed significantly lower (p < 0.05) basal total plasma cortisol than controls. CONCLUSION: The present data validate and substantiate our preliminary evidence for a dysregulation of the HPA axis in patients with FM, marked by mild hypocortisolemia, hyperreactivity of pituitary ACTH release to CRH, and glucocorticoid feedback resistance. Patients with LBP also display hypocortisolemia, but only a tendency toward the disrupted HPA features observed in the patients with FM. We propose that a reduced containment of the stress-response system by corticosteroid hormones is associated with the symptoms of FM.

Dexamethasone concentration in vitreous and serum after oral administration.

PURPOSE: To determine the dexamethasone concentration in vitreous and serum of patients after oral administration of dexamethasone and to compare the results with the concentrations in vitreous and serum found in a previous study with peribulbar injection of 5 mg dexamethasone disodiumphosphate. METHODS: In a prospective study, 54 patients who were scheduled for vitrectomy received 7.5 mg dexamethasone orally at varied time intervals before surgery. A vitreous sample was taken from each patient and serum samples were collected at multiple time points from 32 out of 54 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: Dexamethasone concentrations in serum ranged from 2.5 to 98.1 ng/ml (median, 61.6 ng/ml) between 1 and 3 hours after oral administration of 7.5 mg dexamethasone. Serum concentrations after peribulbar injection of 5 mg dexamethasone disodiumphosphate (containing 3.75 mg dexamethasone) were lower by a factor of 1.5. Concentrations in vitreous ranged from 1.7 to 23.4 ng/ml (median, 5.2 ng/ml) between 4 and 10 hours after oral administration. After peribulbar injection of 5 mg dexamethasone disodiumphosphate, the intravitreal concentrations were 3.9 times higher. CONCLUSIONS: An oral dose of 7.5 mg dexamethasone resulted in an intravitreal corticosteroid concentration with an anti-inflammatory potency that is clearly above physiological level. This concentration, however, is several times lower than is the intravitreal concentration after a peribulbar injection of 5 mg dexamethasone disodiumphosphate, although the two routes of administration resulted in nearly equal dexamethasone concentrations in serum. The higher intravitreal concentration after peribulbar injection is probably caused by diffusion from the serum and additional transscleral diffusion.

Cisplatin combination chemotherapy induces a fall in plasma antioxidants of cancer patients.

BACKGROUND: Antioxidants protect the body against cellular oxidative damage and thus some of the adverse effects induced by cisplatin and other cytostatic drugs. PATIENTS AND METHODS: The effect of cisplatin-combination chemotherapy on concentrations of plasma antioxidants was studied in 36 cancer patients, including osteosarcoma and testicular carcinoma patients. RESULTS: Eight to 15 days after the start of each cytostatic drug infusion concentrations of various plasma antioxidants were measured and compared to pretreatment values: vitamin C and E, uric acid and ceruloplasmin levels fell significantly (P < 0.01-0.005) and returned to baseline levels before the start of the next chemotherapy cycle. Levels of the antioxidants bilirubin albumin and the ratio vitamin E/cholesterol + triglycerides measured three weeks after the start of chemotherapy significantly decreased compared to pretreatment levels and remained low thereafter (P < 0.001-0.002). Dietary intake of antioxidants and anthropometric measurements, evaluated in 14 patients did not change during the whole treatment period. CONCLUSIONS: Cisplatin-combination chemotherapy induces a fall in plasma antioxidant levels, that may reflect a failure of the antioxidant defense mechanism against oxidative damage induced by commonly used anticancer drugs. This probably results from consumption of antioxidants caused by chemotherapy induced-oxidative stress as well as renal loss of water-soluble, small molecular weight antioxidants such as uric acid.