Separating parental and treatment contributions to perinatal health after fresh and frozen embryo transfer in assisted reproduction: A cohort study with within-sibship analysis

BackgroundCompared to naturally conceived children, adverse perinatal outcomes are more common among children born after assisted reproductive technology with fresh embryo transfer (fresh-ET) or frozen embryo transfer (frozen-ET). However, most previous studies could not adequately control for family confounding factors such as subfertility. We compared birth size and duration of pregnancy among infants born after fresh-ET or frozen-ET versus natural conception, using a within-sibship design to account for confounding by maternal factors.Methods and findingsThis registry-based cohort study with nationwide data from Denmark (1994–2014), Norway (1988–2015), and Sweden (1988–2015) consisted of 4,510,790 live-born singletons, 4,414,703 from natural conception, 78,095 from fresh-ET, and 17,990 from frozen-ET. We identified 33,056 offspring sibling groups with the same mother, conceived by at least 2 different conception methods. Outcomes were mean birthweight, small and large for gestational age, mean gestational age, preterm (<37 weeks, versus ≥37), and very preterm birth (<32 weeks, versus ≥32). Singletons born after fresh-ET had lower mean birthweight (−51 g, 95% CI −58 to −45, p < 0.001) and increased odds of small for gestational age (odds ratio [OR] 1.20, 95% CI 1.08 to 1.34, p < 0.001), while those born after frozen-ET had higher mean birthweight (82 g, 95% CI 70 to 94, p < 0.001) and increased odds of large for gestational age (OR 1.84, 95% CI 1.56 to 2.17, p < 0.001), compared to naturally conceived siblings. Conventional population analyses gave similar results. Compared to naturally conceived siblings, mean gestational age was lower after fresh-ET (−1.0 days, 95% CI −1.2 to −0.8, p < 0.001), but not after frozen-ET (0.3 days, 95% CI 0.0 to 0.6, p = 0.028). There were increased odds of preterm birth after fresh-ET (OR 1.27, 95% CI 1.17 to 1.37, p < 0.001), and in most models after frozen-ET, versus naturally conceived siblings, with somewhat stronger associations in population analyses. For very preterm birth, population analyses showed increased odds for both fresh-ET (OR 2.03, 95% CI 1.90 to 2.12, p < 0.001) and frozen-ET (OR 1.66, 95% CI 1.42 to 1.94, p < 0.001) compared with natural conception, but results were notably attenuated within siblings (OR 1.18, 95% CI 1.0 to 1.41, p = 0.059, and OR 0.92, 95% CI 0.67 to 1.27, p = 0.6, for fresh-ET and frozen-ET, respectively). Sensitivity analyses in full siblings, in siblings born within 3-year interval, by birth order, and restricting to single embryo transfers and blastocyst transfers were consistent with the main analyses. Main limitations were high proportions of missing data on maternal body mass index and smoking.ConclusionsWe found that infants conceived by fresh-ET had lower birthweight and increased odds of small for gestational age, and those conceived by frozen-ET had higher birthweight and increased odds of large for gestational age. Conception by either fresh-ET or frozen-ET was associated with increased odds of preterm birth. That these findings were observed within siblings, as well as in conventional multivariable population analyses, reduces the likelihood that they are explained by confounding or selection bias.Trial registrationClinicalTrials.gov ISRCTN11780826.

Kjersti Westvik-Johari and co-workers report on perinatal outcomes following fresh and frozen embryo transfer.     

Oligoclonal Band Status in Scandinavian Multiple Sclerosis Patients Is Associated with Specific Genetic Risk Alleles

The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in multiple sclerosis (MS). We applied data from Norwegian, Swedish and Danish (i.e. Scandinavian) MS patients from a genome-wide association study (GWAS) to search for genetic differences in MS relating to OCB status. GWAS data was compared in 1367 OCB positive and 161 OCB negative Scandinavian MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. HLA-DRB1 genotypes were analyzed in a subset of the OCB positive (n = 2781) and OCB negative (n = 292) MS patients and compared to 890 healthy controls. Results from the genome-wide analyses showed that single nucleotide polymorphisms (SNPs) from the HLA complex and six other loci were associated to OCB status. In SNPs selected for replication, combined analyses showed genome-wide significant association for two SNPs in the HLA complex; rs3129871 (p = 5.7×10⁻¹⁵) and rs3817963 (p = 5.7×10⁻¹⁰) correlating with the HLA-DRB1*15 and the HLA-DRB1*04 alleles, respectively. We also found suggestive association to one SNP in the Calsyntenin-2 gene (p = 8.83×10⁻⁷). In HLA-DRB1 analyses HLA-DRB1*15∶01 was a stronger risk factor for OCB positive than OCB negative MS, whereas HLA-DRB1*04∶04 was associated with increased risk of OCB negative MS and reduced risk of OCB positive MS. Protective effects of HLA-DRB1*01∶01 and HLA-DRB1*07∶01 were detected in both groups. The groups were different with regard to age at onset (AAO), MS outcome measures and gender. This study confirms both shared and distinct genetic risk for MS subtypes in the Scandinavian population defined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS with implications for patient management, which need to be further studied.     

Soluble Markers of the Toll-Like Receptor 4 Pathway Differentiate between Active and Latent Tuberculosis and Are Associated with Treatment Responses

Background

Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment.

Methods

Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied.

Results

Plasma levels of sCD14 (p<0.001), MD-2 (p = 0.036) and LPS (p = 0.069) were elevated at baseline in patients with untreated active TB compared to the LTBI group. MD-2 concentrations decreased after 2 weeks of treatment (p = 0.011), while LPS levels decreased after 8 weeks (p = 0.005). In contrast, sCD14 levels increased after 2 weeks (p = 0.047) with a subsequent modest decrease throughout the treatment period. There was no significant difference in concentrations of any of these markers between patients with pulmonary and extrapulmonary TB or between patients with or without symptoms.

Conclusion

Our data suggest that plasma levels of LPS, MD-2 and sCD14 can discriminate between active TB and LTBI. A decline in LPS and MD-2 concentrations was associated with response to anti-TB treatment. The clinical potential of these soluble TLR-4 pathway proteins needs to be further explored.     

The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8⁺/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs.     

Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal β-cell lines

Insulin secretion from pancreatic β-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus-secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 β-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional β-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus-secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1α (hypoxia-inducible factor 1α). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1α target genes in islets from T2D patients.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.     

Natural killer cells act as early responders in an experimental infection with Neospora caninum in calves

The intracellular protozoan parasite Neospora caninum is a cause of abortion and congenital disease in cattle worldwide. We have previously shown that natural killer (NK) cells produce IFN-gamma in response to N. caninum tachyzoites in vitro. This study aimed to investigate the role of NK cells and other cellular immune responses in an experimental N. caninum infection model in calves. Phenotyping of peripheral blood lymphocytes showed a drop in the percentage of NK cells at days 4-6 after i.v. inoculation, followed by an increase in the percentage of both NK cells and CD8+ T cells which peaked at days 11-15. A whole blood flow cytometric assay showed that CD4+ T cells were the major IFN-gamma producing cells, but in the early stages of the infection both NK cells and CD8+ T cells contributed to IFN-gamma production. We also compared the ability of two different N. caninum antigen preparations--sonicated soluble antigens and intact heat-inactivated parasites--to induce proliferation and IFN-gamma production in various cell types. Heat-inactivated tachyzoites induced a 3.7 times greater increase in the number of IFN-gamma producing NK cells compared with sonicated soluble antigens. This indicated the presence of some NK cell-stimulating antigens in the intact tachyzoite that were absent from the sonicated soluble antigens. The heat-inactivated whole tachyzoites also inhibited gammadelta T cell proliferation while the soluble antigens from N. caninum did not. We believe this is the first time NK cells have been demonstrated to be early responders in N. caninum infection in calves.     

<Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactones are degraded via an amidolytic activity in <Emphasis Type="Italic">Comamonas</Emphasis> sp. strain D1

Abtsract Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.