Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Bone marrow-derived hematopoietic stem and progenitor cells infiltrate allogeneic and syngeneic transplants.

Lineage (CD3e, CD11b, GR1, B220 and Ly-76) negative hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) infiltrate islet allografts within 24 h posttransplantation. In fact, lineage(negative) Sca-1(+) cKit(+) ("LSK") cells, a classic signature for HSCs, were also detected among these graft infiltrating cells. Lineage negative graft infiltrating cells are functionally multi-potential as determined by a standard competitive bone marrow transplant (BMT) assay. By 3 months post-BMT, both CD45.1 congenic, lineage negative HSCs/HPCs and classic "LSK" HSCs purified from islet allograft infiltrating cells, differentiate and repopulate multiple mature blood cell phenotypes in peripheral blood, lymph nodes, spleen, bone marrow and thymus of CD45.2 hosts. Interestingly, "LSK" HSCs also rapidly infiltrate syngeneic islet transplants as well as allogeneic cardiac transplants and sham surgery sites. It seems likely that an inflammatory response, not an adaptive immune response to allo-antigen, is responsible for the rapid infiltration of islet and cardiac transplants by biologically active HSCs/HPCs. The pattern of hematopoietic differentiation obtained from graft infiltrating HSCs/HPCs, cells that are recovered from inflammatory sites, as noted in the competitive BMT assay, is not precisely the same as that of intramedullary HSCs. This does not refute the obvious multi-lineage potential of graft infiltrating HSCs/HPCs.

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1.

Exposure of keratinocytes (KC) to ultraviolet (UV) radiation results in the initiation of apoptosis, a protective mechanism that eliminates cells harboring irreparable DNA damage. Hence, a manipulation of UV-induced apoptosis may significantly influence photocarcinogenesis. We have discovered that the aryl hydrocarbon receptor (AHR), a key regulator of drug metabolism and an UVB-sensitive transcription factor, serves an anti-apoptotic function in UVB-irradiated human KC. Chemical and shRNA-mediated inhibition of AHR signaling sensitized KC to UVB-induced apoptosis by decreasing the expression of E2F1 and its target gene checkpoint kinase 1 (CHK1). The decreased expression of these cell-cycle regulators was due to an enhanced expression of p27(KIP1) and an associated decrease in phosphorylation of both cyclin-dependent kinase 2 and its substrate molecule retinoblastoma protein. The subsequent inhibition of E2F1 autoregulation and downstream CHK1 expression resulted in an enhanced susceptibility of damaged cells to undergo apoptosis. Accordingly, ectopic overexpression of either E2F1 or CHK1 in AHR-knockdown KC attenuated the observed sensitization to UVB-induced apoptosis. Using an AHR-knockout SKH-1 hairless mouse model, we next demonstrated the physiological relevance of the anti-apoptotic function of AHR. In contrast to their AHR-proficient littermates, the constitutive expression of E2F1 and CHK1 was significantly reduced in the skin of AHR-knockout mice. Accordingly, a single exposure of the animals to UVB resulted in an enhanced cleavage of caspase-3 in the skin of AHR-knockout mice. These results identify for the first time the AHR-E2F1-CHK1 axis as a novel anti-apoptotic pathway in KC, which may represent a suitable target for chemoprevention of non-melanoma skin cancer.

Final ART success rates: a 10 years survey.

BACKGROUND: Cumulative pregnancy rates (CPRs) and live birth rates (CLBRs) are much better indicators of success in IVF programmes than cross-sectional figures per cycle or embryo transfer. They allow a better estimation of patient's chances of having a child and enable comparisons between centres and treatment strategies. METHODS: A 10 year cohort study of patients undergoing their first assisted reproductive technique cycle was conducted. Patients were followed until live birth or discontinuation of treatment. All IVF and ICSI cycles and cryo-cycles with embryos derived from frozen pronuclear stage oocytes were included. The CPR and CLBR were estimated using the Kaplan-Meier method for both the number of treatment cycles and transferred embryos. The analysis assumed that couples who did not return for subsequent treatment cycles would have had the same chance of success as those who had continued treatment. RESULTS: A total of 3011 women treated between 1998 and 2007 were included, and 2068 children were born; women already with a live birth re-entered the analysis as a 'new patient'. For 3394 'patients under observation' with 8048 cycles, the CLBR was 52% after 3 cycles (the median number of cycles per patient), 72% after 6 cycles and 85% after 12 cycles. A CLBR of ∼ 50% was achieved for patients aged under 40 years, after the cumulative transfer of six embryos. The mean live birth rate from one fresh cycle and its subsequent cryo-cycle(s) was 33%. Our analysis also shows that ART can reach natural fertility rates but not exceed them. CONCLUSIONS: Most couples with infertility problems can be treated successfully if they continue treatment. Thereby ART can reach natural fertility rates. Even with the restrictions in place as a result of the German Embryo Protection Law, CLBR reach internationally comparable levels.

Isolated CD39 expression on CD4+ T cells denotes both regulatory and memory populations.

Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.

Relevance of anti-Mullerian hormone measurement in a routine IVF program.

BACKGROUND: Diminished ovarian reserve has become a major cause of infertility. Anti-Mullerian hormone (AMH) seems to be a promising candidate to assess ovarian reserve and predict the response to controlled ovarian hyperstimulation (COH). This prospective study was conducted to evaluate the relevance of AMH in a routine IVF program. METHODS: Three hundred and sixteen patients were prospectively enrolled to enter their first IVF/ICSI-cycle. Age, FSH-, inhibin B- and AMH-levels and their predictive values for ovarian response and clinical pregnancy rate were compared by discriminant analyses. RESULTS: A total of 132 oocyte retrievals were performed. A calculated cut-off level < or =1.26 ng/ml AMH alone detected poor responders (< or =4 oocytes) with a sensitivity of 97%, and there was a 98% correct prediction of normal response in COH if levels were above this threshold. With levels <0.5 ng/ml, a correct prediction of very poor response (< or =2 oocytes) was possible in 88% of cases. Levels of AMH > or =0.5 ng/ml were not significantly correlated with clinical pregnancy rates. CONCLUSIONS: AMH is a predictor of ovarian response and suitable for screening. Levels < or =1.26 ng/ml are highly predictive of reduced ovarian reserve and should be confirmed by a second line antral follicle count. Measurement of AMH supports clinical decisions, but alone it is not a suitable predictor of IVF success.

Hematologic and blood biochemical variables of captive chimpanzees: cross-sectional and longitudinal analyses.

Hematologic and blood biochemical variables are of great importance in medical and veterinary practice. In addition, these analytes may have significance as potential biomarkers of aging. Previous reports on normative values of these variables in the chimpanzees are based on cross-sectional studies that did not include individuals of advanced age. To address this omission, we performed cross-sectional and longitudinal analyses of hematologic and blood biochemical data collected from chimpanzees over a 9-year period. One-hundred forty-six females and 106 males of ages representing the entire life span of the species were studied. We derived normative cross-sectional values of 14 commonly measured hematologic and 20 blood biochemical variables, which should provide a useful reference for clinical blood studies in chimpanzees. In addition, we found in a cross-sectional regression analysis of our data that most analytes varied significantly between males and females, and that they varied markedly with age. Most variables had year-to-year consistency within the same individuals, as indicated by statistically significant intra-year correlation coefficients. Finally, we performed a longitudinal analysis of the analytes in chimpanzees by calculating the slopes and intercepts of the best-fitting trend line for each individual. The resulting slopes were analyzed by sex and by decade of age of subjects to determine whether trends were consistent. Consistent trends detected in the longitudinal analysis were usually restricted to the first decade of life, and thus represented maturational processes. The overall lack of within-animal trends covering all or most of the period from early adulthood through old age in this 9-year study suggests that a longer period of follow-up than used here may be required to document senescence-related changes.

The human B cell response to IL-13 is dependent on cellular phenotype as well as mode of activation.

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Ralpha1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Ralpha1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Ralpha1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.

Brain weight throughout the life span of the chimpanzee.

Studies on human postmortem material report lower brain weights in older than in younger cohorts, whereas there is no apparent change with age in the rhesus monkey. In view of these contrasting results, we examined the pattern of brain weight across the life span in the chimpanzee, one of the closest biological relatives of humans. To place the study in context of the empirical life expectancy of the chimpanzee, we first performed a survival analysis on data from 275 chimpanzees that were maintained in the colony of the Yerkes Primate Center. The survival analysis revealed the maximum life spans of female and male chimpanzees to be about 59 and 45 years, respectively. We examined fresh brain weights from 76 chimpanzees ranging in age from birth to 59.4 years of age. The brains were taken from 9 infants (birth to 1 year of age), 25 juveniles (1-7 years), 13 adolescents (7-15 years), 21 young adults (15-30 years), and 8 old adults (over 30 years). Adult brain weight was achieved by the age of 7 years. The adolescent and young adult chimpanzees had the largest brain weights; in these two age groups combined, the mean brain weight (+/- standard deviation) was 368.1 g (+/-37.3) for females (n = 17) and 405.6 g (+/-39.4) for males (n = 17). This sex difference was statistically significant (P < 0.01). Simple linear regression performed on the combined material from females and males aged 7 years and older revealed a decline in brain weight with advancing age of 1.1 g/year (P < 0.05). When the effect of sex on brain weight was statistically controlled for, the loss of brain weight with age was 0.9 g/year (P = 0.07). These results suggest that brain weight declines moderately with age in the chimpanzee as it does in humans.

Brain weight does not decrease with age in adult rhesus monkeys.

Cross-sectional studies on adult human autopsy material have shown that younger cohorts have heavier brains than older groups. We sought to determine whether a similar pattern is present in the rhesus monkey, a species that serves as a useful model of human brain and cognitive aging. Data were obtained from necropsies of 399 rhesus monkeys (180 females; 219 males), of ages covering the entire adult lifespan of this species. In addition to fresh brain weight, variables considered were age, sex, body weight, heart weight, identity of the prosector, and circumstance of death. Initial bivariate analyses revealed a significant sex difference in brain weight (mean for males: 96.1 g; for females: 86.1 g; p < 0.001), as well as significant correlations of brain weight with body weight (r = 0.20, p < 0.01 for females; r = 0.27, p < 0.001 for males), and heart weight (r = 0.27, p < 0.001 for females; r = 0.38, p < 0.001 for males). Identity of prosector, circumstance of death, and age were not significantly related to brain weight in bivariate analyses. Multiple linear regression, controlling for possible confounding effects of body weight and sex, also suggested that brain weight is stable throughout adulthood in the rhesus monkey.

Distribution of muscarinic cholinergic receptor proteins m1 to m4 in area 17 of normal and monocularly deprived rhesus monkeys.

Antibodies to muscarinic cholinergic receptor proteins m1 to m4 were used in striate cortex tissue of normal rhesus monkeys to determine the laminar distribution of these proteins with special attention to geniculorecipient layers. The normal patterns were compared to those of monkeys whose ocular dominance system had been altered by visual deprivation. In normal monkeys, immunoreactivity of all four proteins was localized in complex laminar patterns; m1 was densest in layers 2, 3, and 6, followed by layer 5. In contrast, m2 reactivity was densest in lower layer 4C and in 4A; the latter exhibited a honeycomb pattern. Layers 2 and 3 displayed alternating dense and light regions; this pattern was complementary to that of cytochrome oxidase (CytOx). Laminar immunoreactivity for the m3 receptor was similar to the CytOx pattern, including a honeycomb in 4A and a pattern of alternating darker and lighter patches in layers 2/3. Antibody to m4 reacted most densely with layers 1, 2, 3, and 5, layers 2 and 3 exhibited alternating dark and light regions, and layer 4A had a faint honeycomb. Layer 4C was the lightest band. The differential distribution of these four muscarinic receptor subtypes suggests distinct roles in cholinergic modulation of visual processing in the primate striate cortex. Furthermore, all four muscarinic receptors appear to be insensitive to elimination of visual input via monocular occlusion from birth, to deprivation of pattern vision in one eye during a specific time period in adulthood, and to long-term retinal injury.

Markers of platelet activation and oxidant stress in atherothrombotic disease.

Several new approaches to the study of platelet activation have been developed. Logically, these should be combined with novel indices of coagulant function (60,61) to select rational targets for antithrombotic drugs. They may also be invaluable in dose-finding, which has been a particular weakness in this area of drug development (62,63). While activation of platelets and the coagulation cascade are virtually simultaneous events, markers of the atherosclerosis are also artificially segregated from those of the complicating thrombotic process. Oxidant stress has been implicated in both platelet activation (64) and atherogenesis (65), yet our ability to study this system has been so constrained that we are unsure of appropriate doses of antioxidant vitamins. Novel approaches to this problem promise the ability to study oxidative modification of proteins (46,66,67), lipids (57) and DNA (45,68) in clinical studies.

beta-Amyloid (A beta) deposition in the brains of aged orangutans.

While aged monkeys of several species show cerebral amyloid deposition in senile plaques and blood vessels similar to that seen in human aging and Alzheimer's disease (AD), studies of great apes have been limited. Using histological and immunohistochemical methods, we examined the brains of four orangutans aged 10, 28, 31, and 36 years. We encountered sparse beta-amyloid (A beta)-immunoreactive, silver-negative plaque-like structures in the brains of the three older apes. The 36-year-old orangutan also evidenced small A beta-positive deposits in subcortical white matter and sparse vascular amyloid deposition, primarily in meningeal vessels. Neurofibrillary tangles were not detected on silver stains or on tau or ubiquitin immunohistochemistry. Many of the A beta-positive plaque-like deposits in the orangutans were apolipoprotein E-immunoreactive, as we have previously reported in aged rhesus monkeys and an aged chimpanzee. Also, paralleling our earlier findings in these nonhuman primates, A beta 40 in plaques was more prominent in the orangutan than is typically seen in human aging, AD, and Down syndrome. These intriguing species differences may provide clues to the mechanisms of amyloid deposition and the development of neuropathologic changes in AD.

Neuropeptide Y in the infundibular nucleus and hypophysis of great apes.

We studied the distribution of neuropeptide Y (NPY) immunoreactivity in the infundibular nucleus and the hypophysis of the chimpanzee, gorilla, and orangutan. Using antibodies developed in rabbit against synthetic porcine NPY, we found numerous NPY-immunoreactive neuronal somata in the infundibular nucleus; this nucleus was also filled with short NPY-positive processes and an abundance of punctate structures that could be indicative of synaptic terminals. Numerous varicose NPY-positive fibers were concentrated in the upper infundibular stem in association with capillary loops of the portal vasculature and with the long portal vessels. Bundles of long varicose fibers ran down the infundibular stem, some appearing to terminate in the lower stem in the vicinity of short portal vessels. The bulbous infundibular process contained only sparsely distributed fibers; they were mostly concentrated near vessels at the border between the infundibular process and the anterior pituitary gland, where the fibers often terminated in a spray-like fashion near blood vessels. No NPY immunoreactivity was seen in the anterior pituitary gland. These results provide anatomical evidence for the release of NPY into the portal vasculature of great apes.

A beta40 is a major form of beta-amyloid in nonhuman primates.

Because aged nonhuman primates show beta-amyloid (A beta) deposition in senile plaques and blood vessels similar to that seen in human aging and AD, we used C-terminal specific antibodies to A beta40 and A beta42 to investigate A beta peptide length in the brains of 11 aged rhesus monkeys and a 59-year-old chimpanzee. In contrast to AD, where the earliest and most prominent form of A beta in senile plaques is A beta42, in the monkey, A beta40-positive plaques predominated. The ratio of A beta40:A beta42-positive plaques averaged 2.08 in the monkey, as compared to a mean ratio of 0.37 in 68 human AD subjects (p < 0.001). A beta40 was also more prominent in the chimpanzee than in humans. Possible explanations for these findings include species differences in the cleavage of A beta from the amyloid precursor protein or in the activity of a putative carboxy peptidase forming A beta40 from A beta42 in situ.

Neurobiological bases of age-related cognitive decline in the rhesus monkey.

The rhesus monkey offers a useful model of normal human aging because when monkeys are tested on a battery of behavioral tasks that can also be used to evaluate cognition in humans, it is found that the monkeys undergo an age-related decline in several domains of cognitive function as do humans. In monkeys these changes begin at about 20 years of age. To determine what gives rise to this cognitive decline, we have examined several parameters in the brains of monkeys. Some parameters do not change with age. Examples of this are the numbers of neurons in the neocortex and hippocampal formation, and the numbers of synapses in the hippocampal formation. Changes in other parameters can be positively correlated with chronological age; examples of this are numbers of neuritic plaques, a decrease in the numbers of neurons in the striatally projecting pars compacta of the substantia nigra, and a decrease in the thickness of layer I in primary visual cortex. But the most interesting changes are those that correlate either with cognitive decline alone, or with both cognitive decline and chronological age. Among these are a breakdown in the integrity of myelin around axons, an overall reduction in the volume of white matter in the cerebral hemispheres, thinning of layer I in area 46 of prefrontal cortex, and decreases in the cell density in cortically projecting brain stem nuclei. To date then, our studies suggest that the cognitive declines evident in the rhesus monkey may be a consequence of changes in layer I and in the integrity of myelinated axons, rather than an age-related loss of cortical neurons or synapses, as has long been assumed.

Preservation into old age of synaptic number and size in the supragranular layer of the dentate gyrus in rhesus monkeys.

In order to determine whether there are age-related changes in the supragranular layer of the dentate gyrus of the rhesus monkey, we examined this layer in monkeys 4-35 years of age, spanning the entire range of adulthood of this species. Electron microscopic analyses were conducted to determine whether there is an age-related change in the number of synapsing axon terminals, in the cross-sectional area of these terminals, or in the length of the postsynaptic density at the synaptic junction. Only asymmetrical synapses in the anterior dentate gyrus were evaluated. In a subset of our monkeys (n = 6, ages 4-31 years), we compared three different approaches to the estimation of synaptic density: (1) the conventional profile method, in which synapse numbers are expressed per unit area of the examined tissue section; (2) the empirical formula of Colonnier and Beaulieu for converting areal densities into number of synapses per unit volume, and (3) the 'disector' method, a stereological approach to the estimation of the number of synapses per unit volume that makes no assumption about the shape of the objects. Data are presented validating the small-fold method of estimating section thickness for the disector. The three methods were highly intercorrelated (rs approximately 0.89), and none of the methods revealed an age-related loss of synapses. Analysis of the thickness of the dentate gyrus molecular layer suggests that the reference volume in which these synapses were counted dose not change with age. In addition, the conventional profile method showed no age-related change in the number of axodendritic or axospinous synapses, the cross-sectional area of the synapsing terminals, or the length of the postsynaptic densities of synapses. Together, these data suggest a remarkable age-related preservation of synapses in the normal aging monkey.

Sources of subcortical afferents to the macaque's dorsal lateral geniculate nucleus.

BACKGROUND: The dorsal lateral geniculate nucleus (dLGN) is the thalamic region responsible for transmitting retina signals to cortex. Brainstem pathways to this nucleus have been described in several species and are believed to control the retinocortical pathway depending on the state of the animal (awake, asleep, drowsy, etc.). The purpose of this study was to determine all of the subcortical sources of afferents to the dLGN in a higher primate, the macaque monkey, whose visual system is similar to that of humans. METHODS: Injections of horseradish peroxidase (HRP), with or without conjugation to wheat germ agglutinin, were made into the dLGNs of seven macaque monkeys, followed by perfusion, brain sectioning, and analyses of neurons in the brainstem, thalamus, and hypothalamus that contained the retrogradely transported marker. RESULTS: The reticular nucleus of the thalamus, pedunculopontine nucleus, parabigeminal nucleus, pretectal nucleus of the optic tract, superior colliculus, dorsal raphe nucleus, and tuberomammillary region of the hypothalamus contained many retrogradely labeled neurons ipsilateral to the injections. In the contralateral brainstem, HRP-labeled cells were found only in the pedunculopontine nucleus, nucleus of the optic tract, and dorsal raphe nucleus. The number of labeled neurons on the contralateral side was about one-half of that in corresponding ipsilateral nuclei. The locus coeruleus contained no labeled neurons in four of the macaques that had injections limited to the dLGN. CONCLUSION: There are seven subcortical regions that send afferents to the dLGNs of macaque monkeys. Except for the locus coeruleus, these are the same as observed for other species, such as the cat and rat, and indicate the possible sources of subcortical control over the dLGNs of humans.