Exogenous gonadotropins have little impact on follicular but considerable effect on serum cytokine concentrations – a comparison between Natural Cycle and stimulated IVF using a multiplexed assay platform

Introduction: Throughout follicular growth and subsequent corpus luteum formation the leukocyte number increases and follicular vascularisation changes. These processes are enhanced under exogenous stimulation with gonadotropins. Cytokines released by leukocytes contribute to further recruitment and vascularisation of the follicle, and they play an important role in regulating ovarian steroidogenesis by influencing theca and granulosa–lutein cell function. Changes in cytokine and vascular endothelial growth factor (VEGF) concentrations in the ovary as a consequence of gonadotropin stimulation may negatively influence oocyte quality. In this project we have compared the intrafollicular production of inflammatory cytokines and growth factors between natural IVF cycles (NC) and classical, gonadotropin-stimulated IVF cycles (gsIVF). Material and Methods: Serum on the day of oocyte retrieval and follicular fluid (FF) were collected in 37 NC and 39 gsIVF cycles. Thirteen women within this population underwent one NC and one gsIVF cycle each. A total of 14 cytokines from Bio-Plex panels I and II were determined in matched serum and FF samples using Luminex xMAP technology on the Bio-Plex(R) platform, using the serum protocol. Results: Tumour necrosis factor-alpha, RANTES, eotaxin and interferon-gamma-induced protein-10 levels were lower in FF than in serum, and thus not further investigated. Interleukin (IL)-6, -8, -10, -15, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median concentrations in FF than in serum, indicating possible ovarian production. Moreover, most of these showed higher evels in the gsIVF than in the NC groups in the serum, but not in the follicular fluid. IL-8 was reduced in gsIVF cycles. Conclusion: The fact that serum but not FF levels of the studied cytokines were higher in the stimulated than in the natural cycles can be attributed to the increased number of active follicles present after controlled ovarian stimulation.

Exogenous gonadotropins do not increase the blood-follicular transportation capacity of extra-ovarian hormones such as prolactin and cortisol.

BACKGROUNDS In vitro fertilization involves high dosage gonadotropin stimulation, which apparently has some negative impact on follicular endocrine function. As chorionic gonadotropin stimulation has been shown to increase the blood-follicular permeability in animal models, this raises the question if such an effect also applies to gonadotropins in humans, possibly affecting the endocrine follicular milieu. FINDINGS Follicular fluid and serum were collected at the time of follicular aspiration in in vitro fertilisation without (Natural cycle IVF, n = 24) and with (conventional gonadotropin stimulated IVF, n = 31) gonadotropin stimulation. The concentration of the extra-ovarian hormones prolactin and cortisol were analysed by immunoassays. RESULTS Median serum prolactin and cortisol concentrations were 12.3 ng/mL and 399 nmol/L without versus 32.2 ng/mL and 623 nmol/L with gonadotropin stimulation. The corresponding concentrations in follicular fluid were 20.6 ng/mL and 445 nmol/L versus 28.8 ng/ml and 456 nmol/L for prolactin and cortisol. As a consequence, mean follicular fluid:serum ratios were significantly reduced under gonadotropin stimulation (prolactin p = 0.0138, cortisol p = 0.0001). As an enhanced blood-follicular permeability and transportation, induced by gonadotropin stimulation, would result in increased instead of decreased follicular fluid:serum ratios as found in this study, it can be assumed that this does not affect extra-ovarian protein and steroid hormones as illustrated by prolactin and cortisol. CONCLUSIONS The model of serum follicular fluid:serum ratio of hormones, produced outside the ovaries, did not reveal a gonadotropin induced increased blood-follicular transportation capacity. Therefore it can be assumed that the effect of gonadotropins on follicular endocrine function is not due to an increased ovarian permeability of extra-ovarian hormones.

Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L.

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post-clipping. Samples were prepared for histopathological examination to study the course of re-epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post-clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post-clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.

Effect of temperature and diet on wound healing in Atlantic salmon (Salmo salar L.).

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout (Salmo trutta)

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected 'Candidatus Piscichlamydia salmonis' and 'Candidatus Clavochlamydia salmonicola' infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PCR.

Pulmonary hypertension in high-altitude dwellers: novel mechanisms, unsuspected predisposing factors

Studies of high-altitude populations, and in particular of maladapted subgroups, may provide important insight into underlying mechanisms involved in the pathogenesis of hypoxemia-related disease states in general. Over the past decade, studies involving short-term hypoxic exposure have greatly advanced our knowledge regarding underlying mechanisms and predisposing events of hypoxic pulmonary hypertension. Studies in high altitude pulmonary edema (HAPE)-prone subjects, a condition characterized by exaggerated hypoxic pulmonary hypertension, have provided evidence for the central role of pulmonary vascular endothelial and respiratory epithelial nitric oxide (NO) for pulmonary artery pressure homeostasis. More recently, it has been shown that pathological events during the perinatal period (possibly by impairing pulmonary NO synthesis), predispose to exaggerated hypoxic pulmonary hypertension later in life. In an attempt to translate some of this new knowledge to the understanding of underlying mechanisms and predisposing events of chronic hypoxic pulmonary hypertension, we have recently initiated a series of studies among high-risk subpopulations (experiments of nature) of high-altitude dwellers. These studies have allowed to identify novel risk factors and underlying mechanisms that may predispose to sustained hypoxic pulmonary hypertension. The aim of this article is to briefly review this new data, and demonstrate that insufficient NO synthesis/bioavailability, possibly related in part to augmented oxidative stress, may represent an important underlying mechanism predisposing to pulmonary hypertension in high-altitude dwellers.

Whole-body MRI of multiple myeloma: comparison of different MRI sequences in assessment of different growth patterns

PURPOSE: To determine sensitivity, specificity and inter-observer variability of different whole-body MRI (WB-MRI) sequences in patients with multiple myeloma (MM). METHODS AND MATERIALS: WB-MRI using a 1.5T MRI scanner was performed in 23 consecutive patients (13 males, 10 females; mean age 63+/-12 years) with histologically proven MM. All patients were clinically classified according to infiltration (low-grade, n=7; intermediate-grade, n=7; high-grade, n=9) and to the staging system of Durie and Salmon PLUS (stage I, n=12; stage II, n=4; stage III, n=7). The control group consisted of 36 individuals without malignancy (25 males, 11 females; mean age 57+/-13 years). Two observers independently evaluated the following WB-MRI sequences: T1w-TSE (T1), T2w-TIRM (T2), and the combination of both sequences, including a contrast-enhanced T1w-TSE with fat-saturation (T1+/-CE/T2). They had to determine growth patterns (focal and/or diffuse) and the MRI sequence that provided the highest confidence level in depicting the MM lesions. Results were calculated on a per-patient basis. RESULTS: Visual detection of MM was as follows: T1, 65% (sensitivity)/85% (specificity); T2, 76%/81%; T1+/-CE/T2, 67%/88%. Inter-observer variability was as follows: T1, 0.3; T2, 0.55; T1+/-CE/T2, 0.55. Sensitivity improved depending on infiltration grade (T1: 1=60%; 2=36%; 3=83%; T2: 1=70%; 2=71%; 3=89%; T1+/-CE/T2: 1=50%; 2=50%; 3=89%) and clinical stage (T1: 1=58%; 2=63%; 3=79%; T2: 1=58%; 2=88%; 3=100%; T1+/-CE/T2: 1=50%; 2=63%; 3=100%). T2w-TIRM sequences achieved the best reliability in depicting the MM lesions (65% in the mean of both readers). CONCLUSIONS: T2w-TIRM sequences achieved the highest level of sensitivity and best reliability, and thus might be valuable for initial assessment of MM. For an exact staging and grading the examination protocol should encompass unenhanced and enhanced T1w-MRI sequences, in addition to T2w-TIRM.

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

Population transcriptomics of life-history variation in the genus Salmo

In this study, we demonstrate the power of applying complementary DNA (cDNA) microarray technology to identifying candidate loci that exhibit subtle differences in expression levels associated with a complex trait in natural populations of a nonmodel organism. Using a highly replicated experimental design involving 180 cDNA microarray experiments, we measured gene-expression levels from 1098 transcript probes in 90 individuals originating from six brown trout (Salmo trutta) and one Atlantic salmon (Salmo salar) population, which follow either a migratory or a sedentary life history. We identified several candidate genes associated with preparatory adaptations to different life histories in salmonids, including genes encoding for transaldolase 1, constitutive heat-shock protein HSC70-1 and endozepine. Some of these genes clustered into functional groups, providing insight into the physiological pathways potentially involved in the expression of life-history related phenotypic differences. Such differences included the down-regulation of genes involved in the respiratory system of future migratory individuals. In addition, we used linear discriminant analysis to identify a set of 12 genes that correctly classified immature individuals as migratory or sedentary with high accuracy. Using the expression levels of these 12 genes, 17 out of 18 individuals used for cross-validation were correctly assigned to their respective life-history phenotype. Finally, we found various candidate genes associated with physiological changes that are likely to be involved in preadaptations to seawater in anadromous populations of the genus Salmo, one of which was identified to encode for nucleophosmin 1. Our findings thus provide new molecular insights into salmonid life-history variation, opening new perspectives in the study of this complex trait.

Ovarian stimulation to cryopreserve fertilized oocytes in cancer patients can be started in the luteal phase

OBJECTIVE: To analyze if oocytes can be obtained in all patients before cancer treatment within 2 weeks by initiating ovarian stimulation during the follicular or luteal phase. DESIGN: Prospective controlled multicenter trial. SETTING: Four university-based centers. PATIENT(S): Forty cancer patients before chemotherapy. INTERVENTION(S): Twenty-eight patients were stimulated with gonadotropins in the follicular phase (group I). In 12 patients (group II), ovarian stimulation was initiated in the luteal phase, and these received GnRH antagonists and recombinant FSH. In 14 patients, 143 oocytes were further processed for fertilization by intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Number of oocytes aspirated after ovarian stimulation, cumulative FSH/hMG dosage, viability and maturity of oocytes, and fertilization rate by ICSI. RESULT(S): Patients in group I (age 27.6 +/- 4.9 yrs) were stimulated on average for 10.6 days, and patients in group II (age 31.2 +/- 5.7 yrs) for 11.4 days. Total amount of FSH was on average 2,255 IU (I) and 2,720 IU (II) per patient. Average and median numbers of aspirated oocytes were, respectively, 13.1 and 11.5 (I) versus 10.0 and 8.5 (II); 83.7% (I) and 80.4% (II) of the oocytes were mature and viable and could be treated by ICSI. Fertilization rate was 61.0% (I) versus 75.6% (II). CONCLUSION(S): This pilot study suggests that oocytes can be obtained before cancer treatment efficiently irrespective of the phase of the menstrual cycle.

Effects of nasal calcitonin on bone mineral density following parathyroidectomy in patients with primary hyperparathyroidism

To investigate whether nasal salmon calcitonin (CT; 200 U/day) given in addition to calcium helps to restore the bone mass after parathyroidectomy (PTX) in patients with primary hyperparathyroidism (PHPT).

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.